U.S. patent number 8,557,780 [Application Number 13/098,393] was granted by the patent office on 2013-10-15 for monomethylvaline compounds capable of conjugation to ligands.
This patent grant is currently assigned to Seattle Genetics, Inc.. The grantee listed for this patent is Svetlana O. Doronina, Peter D. Senter, Brian E. Toki. Invention is credited to Svetlana O. Doronina, Peter D. Senter, Brian E. Toki.
United States Patent |
8,557,780 |
Doronina , et al. |
October 15, 2013 |
Monomethylvaline compounds capable of conjugation to ligands
Abstract
Auristatin peptides, including MeVal-Val-Dil-Dap-Norephedrine
(MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), were prepared and attached
to Ligands through various linkers, including
maleimidocaproyl-val-cit-PAB. The resulting ligand drug conjugates
were active in vitro and in vivo.
Inventors: |
Doronina; Svetlana O.
(Snohomish, WA), Senter; Peter D. (Seattle, WA), Toki;
Brian E. (Shoreline, WA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Doronina; Svetlana O.
Senter; Peter D.
Toki; Brian E. |
Snohomish
Seattle
Shoreline |
WA
WA
WA |
US
US
US |
|
|
Assignee: |
Seattle Genetics, Inc.
(Bothell, WA)
|
Family
ID: |
34916511 |
Appl.
No.: |
13/098,393 |
Filed: |
April 29, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120003248 A1 |
Jan 5, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11833961 |
Aug 3, 2007 |
7964567 |
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10983340 |
Nov 5, 2004 |
7498298 |
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60622455 |
Oct 27, 2004 |
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60598899 |
Aug 4, 2004 |
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60557116 |
Mar 26, 2004 |
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60518534 |
Nov 6, 2003 |
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Current U.S.
Class: |
514/19.3;
530/330; 514/21.8 |
Current CPC
Class: |
A61K
47/6803 (20170801); A61P 43/00 (20180101); A61K
47/6855 (20170801); A61P 31/04 (20180101); A61P
37/06 (20180101); A61P 35/02 (20180101); C07K
16/32 (20130101); A61P 37/00 (20180101); A61P
31/12 (20180101); A61K 47/6851 (20170801); A61K
47/6889 (20170801); A61P 35/00 (20180101); A61P
31/00 (20180101); C07K 7/02 (20130101); A61P
37/02 (20180101); A61K 47/6811 (20170801); A61K
47/6849 (20170801); Y02A 50/30 (20180101); C07K
2317/24 (20130101); A61K 38/00 (20130101); A61K
2039/505 (20130101); Y10T 428/13 (20150115) |
Current International
Class: |
A61K
38/00 (20060101); C07K 7/02 (20060101); C07K
17/06 (20060101); C07K 17/02 (20060101); C07K
5/027 (20060101); A61K 38/08 (20060101); A61K
38/04 (20060101); A61P 35/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
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2004 213053 |
|
Sep 2004 |
|
AU |
|
2114156 |
|
Jul 1994 |
|
CA |
|
0 598 129 |
|
May 1994 |
|
EP |
|
0 603 735 |
|
Jun 1994 |
|
EP |
|
0 695 757 |
|
Feb 1996 |
|
EP |
|
0 695 758 |
|
Feb 1996 |
|
EP |
|
0 695 759 |
|
Feb 1996 |
|
EP |
|
06-234790 |
|
Aug 1994 |
|
JP |
|
8-59693 |
|
Mar 1996 |
|
JP |
|
09-77791 |
|
Mar 1997 |
|
JP |
|
WO 93/03054 |
|
Feb 1993 |
|
WO |
|
WO 95/09864 |
|
Apr 1995 |
|
WO |
|
WO 96/14856 |
|
May 1996 |
|
WO |
|
WO 96/22384 |
|
Jul 1996 |
|
WO |
|
WO 96/33212 |
|
Oct 1996 |
|
WO |
|
WO 97/25068 |
|
Jul 1997 |
|
WO |
|
WO 99/35164 |
|
Jul 1999 |
|
WO |
|
WO 01/18032 |
|
Mar 2001 |
|
WO |
|
WO 01/40276 |
|
Jun 2001 |
|
WO |
|
WO 01/57188 |
|
Aug 2001 |
|
WO |
|
WO 02/43661 |
|
Jun 2002 |
|
WO |
|
WO 02/43661 |
|
Jun 2002 |
|
WO |
|
WO 02/088172 |
|
Nov 2002 |
|
WO |
|
WO 03/008378 |
|
Jan 2003 |
|
WO |
|
WO 03/022995 |
|
Mar 2003 |
|
WO |
|
WO 03/024392 |
|
Mar 2003 |
|
WO |
|
WO 03/034903 |
|
May 2003 |
|
WO |
|
WO 03/043583 |
|
May 2003 |
|
WO |
|
WO 03/105758 |
|
Dec 2003 |
|
WO |
|
WO 04/001004 |
|
Dec 2003 |
|
WO |
|
WO 2004/010957 |
|
Feb 2004 |
|
WO |
|
WO 2004/016225 |
|
Feb 2004 |
|
WO |
|
WO 2004/032828 |
|
Apr 2004 |
|
WO |
|
WO 2004/045516 |
|
Jun 2004 |
|
WO |
|
WO 2004/073656 |
|
Sep 2004 |
|
WO |
|
WO 2005/084390 |
|
Sep 2005 |
|
WO |
|
2006/034488 |
|
Mar 2006 |
|
WO |
|
WO 2006/071441 |
|
Jul 2006 |
|
WO |
|
2006/083936 |
|
Aug 2006 |
|
WO |
|
2007/001851 |
|
Jan 2007 |
|
WO |
|
WO 2007/030642 |
|
Mar 2007 |
|
WO |
|
WO 2007/059082 |
|
May 2007 |
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WO |
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WO 2007/062138 |
|
May 2007 |
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WO |
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2007/109567 |
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Sep 2007 |
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WO |
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Other References
Hassan et al., "Anti-tumor activity of K1-LysPE38QQR, an
immunotoxin targeting mesothelin, a cell-surface antigen
overexpressed in ovarian cancer and malignant mesothelioma,"
Journal of Immunotherapy, 2000, vol. 23, No. 4, pp. 473-479. cited
by applicant .
Sweet et al., "Daunorubicin conjugated to a monoclonal anti-CA125
antibody selectively kills human ovarian cancer cells," Gynecologic
Oncology, 1989, vol. 34, No. 3, pp. 305-311. cited by applicant
.
Alley et al., "Controlling the location of drug attachment in
antibody-drug conjugates," Proceedings of the AACR, vol. 45,
abstract # 627 (2004). cited by applicant .
Afar et al., "Preclinical validation of anti-TMEFF2-auristatin
E-conjugated antibodies in the treatment of prostate cancer,"
Molecular Cancer Therapeutics, 3(8):921-932 (2004). cited by
applicant .
Bhaskar et al., "E-Selectin Up-Regulation Allows for Targeted Drug
Delivery in Prostate Cancer," Cancer Research, 63:6387-6394 (2003).
cited by applicant .
Carl, P.L. et al., "A novel connector linkage applicable in prodrug
design," Journal of Medicinal Chemistry, May 1981, vol. 24, No. 5,
pp. 479-480. cited by applicant .
Carter, "Improving the Efficacy of Antibody-Based Cancer
Therapies," Nature Reviews 1:118-129 (2001). cited by applicant
.
Dillman, "Monoclonal Antibodies for Treating Cancer," Annals of
Internal Medicine 111:592-603 (1989). cited by applicant .
Doronina et al., "Development of potent monoclonal antibody
auristatin conjugates for cancer therapy," Nature Biotechnology,
21(7):778-784 (2003) + Erratum, Nature Biotechnology, 21(8):941
(2003). cited by applicant .
Doronina et al., "Enhanced Activity of Monomethylauristatin F
through Monoclonal Antibody Delivery: Effects of Linker Technology
on Efficacy and Toxicity," Bioconjugate Chem., 17:114-124 (2006).
cited by applicant .
Doronina et al., "Immunoconjugates comprised of drugs with impaired
cellular permeability: A new approach to targeted therapy,"
SciFinder search result, abstract of paper from 228th ACS National
Meeting held in Philadelphia, PA, Aug. 22-26, 2004. cited by
applicant .
Emery et al., "Humanized monoclonal antibodies for therapeutic
applications," Exp. Opin. Invest. Drugs 3(3):241-251 (1994). cited
by applicant .
Francisco et al. "cAC10-vcMMAE, an anti-CD30-monomethyl auristatin
E conjugate with potent and selective antitumor activity," Blood,
102(4):1458-1465 (2003). cited by applicant .
Gaertner & Offord, "Site-specific attachment of functionalized
poly(ethylene glycol) to the amino terminus of proteins." Bioconj.
Chem. 7(1): 38-44 (1996). cited by applicant .
Garteiz, D.A. et al., "Quantitation of dolastatin-10 using
HPLC/electrospray ionization mass spectrometry: application in a
phase I clinical trial," Cancer Chemother. Pharmacol., 1998, vol.
41, pp. 299-306. cited by applicant .
Genet, J. P., "Recent studies on asymmetric hydrogenation. New
catalysts and synthetic applications in organic synthesis," Pure
Appl. Chem., 74(1):77-83 (2002). cited by applicant .
Hamblett et al., "Effect of drug loading on the pharmacology,
pharmacokinetics, and toxicity of an anti-CD30 antibody-drug
conjugate," Proceedings of the AACR, vol. 45, abstract # 624
(2004). cited by applicant .
Inada et al., "Modification of proteins with polyethylene glycol
derivatives." Methods Enzymol. 242: 65-90 (1994). cited by
applicant .
Jansen, F.K. et al., "Immunotoxins: hybrid molecules combining high
specificity and potent cytotoxicity," Immunol. Rev., 1982, vol. 62,
pp. 185-216. cited by applicant .
Kline et al., "Novel Antitumor Prodrugs Designed for Activation by
Matrix Metalloproteinases-2 and -9," Molecular Pharmaceutics,
1(1):9-22 (2004). cited by applicant .
Klussman et al., "Secondary mAb--vcMMAE conjugates are highly
sensitive reporters of antibody internalization via the lysosome
pathway," Bioconjug Chem., 15(4):765-773 (2004). cited by applicant
.
Mao et al., "EphB2 as a Therapeutic Antibody Drug Target for the
Treatment of Colorectal Cancer," Cancer Research, 64:781-788
(2004). cited by applicant .
Meyer et al., "Recent Advances in Antibody Drug Conjugates for
Cancer Therapy," Annual Reports in Medical Chemistry, 38(chapter
23):229-237 (2003). cited by applicant .
Miyazaki et al., "Synthesis and Antitumor Activity of Novel
Dolastatin 10 Analogs," Chem. Pharm. Bull., 43(10):1706-1718
(1995). cited by applicant .
Natsume et al., "Characterization of the Interaction of TZT-1027, a
Potent Antitumor Agent, with Tubulin," Jpn. J. Cancer, 91:737-747
(2000). cited by applicant .
Petit et al. "Antineoplastic agents 337. Synthesis of dolastatin 10
structural modifications," Anti-Cancer Drug Design, 10:529-544
(1995). cited by applicant .
Petit et al., "Specific Activities of Dolastatin 10 and Peptide
Derivatives against Cryptococcus neoformans," Antimicrobial Agents
and Chemotherapy, 42(11):2961-2965 (1998). cited by applicant .
Petit et al., "A Cobalt--Phosphine Complex Directed Reformatsky
Approach to a Stereospecific Synthesis of the Dolastatin 10 Unit
Dolaproine (Dap)1," J. Org. Chem., 66:8640-8642 (2001). cited by
applicant .
Pettit et al., "The Absolute Configuration and Synthesis of Natural
(-)-Dolastatin 10," J. Am. Chem. Soc., 111:5463-5465 (1989). cited
by applicant .
Pettit et al., "Dolastatins 24. Synthesis of (-)-dolastatin 10.
X-Ray molecular structure of N,N-dimethylvalyl-valyl-dolaisoleuine
tert-butyl ester," J. Chem. Soc. Perkin Trans.1, 5:859-863 (1996).
cited by applicant .
Pettit et al., "Antineoplastic agents 365. Dolastatin 10 SAR
probes," Anticancer Drug Des., 13(4):243-277 (1998). cited by
applicant .
Press Release, "Seattle Genetics, Inc. (SGEN) To Present Advances
In Preclinical Research At American Cancer Research Annual
Meeting," Mar. 24, 2004, downloaded from internet on Aug. 31, 2004.
cited by applicant .
Schoffski et al., "Phase I and pharmacokinetic study of TZT-1027, a
novel synthetic dolastatin 10 derivative, administered as a 1-hour
intravenous infusion every 3 weeks in patients with advanced
refractory cancer," Annals of Oncology, 15:671-679 (2004). cited by
applicant .
Senter et al., "Immunoconjugates comprised of drugs with impaired
cellular permeability: A new approach to targeted therapy,"
Proceedings of the AACR, vol. 45, abstract # 623 (2004). cited by
applicant .
Thornber, "Isosterism and molecular modification in drug design."
Chem. Soc. Rev. 8(4): 563-580 (1979). cited by applicant .
Toki et al., "Protease-Mediated Fragmentation of p-Amidobenzyl
Ethers: A New Strategy for the Activation of Anticancer Prodrugs,"
J. Org. Chem., 67:1866-1872 (2002). cited by applicant .
Vippagunta et al., "Crystalline solids." Adv. Drug Delivery Rev.
48: 3-26 (2001). cited by applicant .
Woyke et al., "Effect of auristatin PHE on microtube integrity and
nuclear localization in Cryptococcus neoformans," Antimicrobial
Agents and Chemotherapy, 46(12):3802-3808 (2003). cited by
applicant .
Woyke et al., "In Vitro Activities and Postantifungal Effects of
the Potent Dolastatin 10 Derivative Auristatin PHE," Antimicrobial
Agents and Chemotherapy, 45(12):3580-3584 (2001). cited by
applicant .
International Search Report of Oct. 2, 2006 for PCT application
PCT/US04/38392, which corresponds to the parent application, U.S.
Appl. No. 10/983,340. cited by applicant .
Written Opinion of Oct. 2, 2006 for PCT application PCT/US04/38392,
which corresponds to the parent application, U.S. Appl. No.
10/983,340. cited by applicant .
Aherne et al., "Antitumour evaluation of dolastatins 10 and 15 and
their measurements in plasma by radioimmunoassay," Cancer
Chemother. Pharmacol., 1996, 38, 225-323. cited by applicant .
Bowman et al. 1950, "N-substituted amino-acids. Part I. A new
method of preparation of dimethylamino-acids,". J. Chem.
Soc.:1342-1351. cited by applicant .
Bradley, C.M. et al., "Limits of Cooperativity in a Structurally
Modular Protein: Response of the Notch Ankyrin Domain to analogous
Alanine Substitutions in Each Request," J. Mol. Biol., 2002, vol.
324, pp. 373-386. cited by applicant .
Carter, P.J. et al., "Antibody-Drug Conjugates for Cancer Therapy,"
The Cancer Journal, May/Jun. 2008, vol. 14, No. 3, pp. 154-169.
cited by applicant .
Dermer, G.B., "Another Anniversary for the War on Cancer,"
Bio/Technology, Mar. 12, 1994, vol. 12, p. 320. cited by applicant
.
Doronina et al., "Errata and Corrigenda," Nature Biotechnology,
2003, 21(8), 941. cited by applicant .
Doronina, S.O. et al., "Potent Monoclonal Antibody-Drug Conjugates:
The Role of Linker Stability in Efficacy, Toxicity and
Specificity," poster #6425 at the annual meeting of the American
Association for Cancer Research, AACR, Toronto, Ontario, CA, Apr.
5-9, 2003. cited by applicant .
Dubowchik, G.M. et al., "Cathepsin B-Sensitive Dipeptide Prodrugs.
2. Models of Anticancer Drugs Paclitaxel (Taxol.RTM.), Mitomycin C
and Doxorubicin," Bioorganic & Medicinal Chemistry Letters,
1998, vol. 8, pp. 3347-3352. cited by applicant .
Engert et al., "Evaluation of Ricin A Chain-containing Immunotoxins
Directed against the CD30 Antigen as Potential Reagents for the
Treatment of Hodgkin's Disease," Cancer Research, 1990, 50, 84-88.
cited by applicant .
Falini et al., "Responses of refractory Hodgkin's disease to
monoclonal anti-CD30 immunotoxin," The Lancet, 1992, 339,
1195-1196. cited by applicant .
Fennell et al., "Effects of the antimitotic natural product
dolastatin 10, and related peptides, on the human malarial parasite
Plasmodium falciparum," Journal of Antimicrobial Chemotherapy,
2003, vol. 51, pp. 833-841. cited by applicant .
Francisco, J.A. et al., "SGN-35, an Anti-CD30 Antibody-Drug
Conjugate with Potent Antitumor Activity," poster #770 at the
annual meeting of the American Association for Cancer Research,
AACR, Toronto, Ontario, CA, Apr. 5-9, 2003. cited by applicant
.
Frisch et al., "Synthesis of Short Polyoxyethylene-Based
Heterobifunctional Cross-Linking Reagents. Application to the
Coupling o Peptides to Liposomes," Bioconjugate Chem., 1996, 7,
180-186. cited by applicant .
Golden, F., "Of mice and men: Don't blame the rodents," Time
Australia, May 18, 1998, 2 pages. cited by applicant .
Gura, T., "Systems for Identifying New Drugs Are Often Faulty,"
Science, Nov. 7, 1997, vol. 278, pp. 1041-1042. cited by applicant
.
Hamann, P.R. et al., "Gemtuzumab Ozogamicin, A Potent and Selective
Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute
Myeloid Leukemia," Bioconjugate Chem., 2002, vol. 13, No. 1, pp.
47-58. cited by applicant .
King et al., "Monoclonal Antibody Conjugates of Doxorubicin
Prepared with Branched Linkers: A Novel Method for Increasing the
Potency of Doxorubicin Immunoconjugates," Bioconjugate Chem., 1999,
10, 279-288. cited by applicant .
King et al., "Monoclonal Antibody Conjugates of Doxorubicin with
Branched Peptide Linkers: Inhibition of Aggregation by
Methoxytriethyleneglycol Chains," J. Med. Chem., 2002, 45,
4336-4343. cited by applicant .
May et al., "Evaluation of Ricin a Chain-Containing Immunotoxins
Directed Against Different Epitopes on the .delta.-Chain of Cell
Surface-Associated IgD on Murine B Cells," The Journal of
Immunology, 1990, 144(9), 3637-3642. cited by applicant .
Meyer, D. et al., "Proteolytic vs. Hydrolytic Released Drug from
Anti-Tumor Immunochemotherapeutic Agents." poster presentation at
Gordon Research Conference on Drug Carriers In Medicine &
Biology, Feb. 24-Mar. 1, 2002, Ventura, California, USA. cited by
applicant .
Mohammad et al., "An Orthotopic Model of Human Pancreatic Cancer in
Severe Combined Immunodeficient Mice: Potential Application for
Preclinical Studies1," Clinical Cancer Research, 1998, 4, 887-894.
cited by applicant .
Pettit et al., 1994, "The Dolastatins. 17. Synthesis of dolaproine
and related diastereoisomers," J. Org. Chem.. 59(21):6287-6295.
cited by applicant .
Pettit et al., 1994, "The Dolastatins. 19. Synthesis of
dolaisoleuine," J. Org. Chem. 59(7):1796-1800. cited by applicant
.
Pettit et al., 1996, "Dolastatins. 23: Stereospecific synthesis of
dolaisoleuine," J. Chem Soc. Perkin. Trans. 1:853-858. cited by
applicant .
Pettit et al., 1996, "The Dolastatins. 18. Stereospecific synthesis
of dolaproine," Synthesis 6:719-725. cited by applicant .
Press et al., "Ricin A-Chain Containing Immunotoxins directed
Against Different Epitopes on the CD2 Molecule Differ in Their
Ability to Kill Normal and Malignant T Cells," The Journal of
Immunology, 1988, 141(12), 4410-4417. cited by applicant .
Rudinger, J., "Characteristics of the amino acids as components of
a peptide hormone sequence," Peptide Hormones, Jun. 1976, pp. 1-6.
cited by applicant .
Schnell et al., "Development of New Ricin A-Chain Immunotoxins With
Potent Anti-Tumor effects Against human Hodgkin Cells In Vitro And
Disseminated Hodgkin Tumors in Scid Mice Using High-Affinity
Monoclonal Antibodies Directed Against The CD30 Antigen," Int. J.
Cancer, 1995, 63, 238-244. cited by applicant .
Senter, P. et al., "Cures and Regressions of Established Tumors
with Monoclonal Antibody-Auristatin E Conjugates." poster
presentation at the annual meeting of the American Association for
Cancer Research, Apr. 6-10, 2002, San Francisco, California, USA.
cited by applicant .
Shioiri et al., 1993, "Stereoselective synthesis of dolastatin 10
and its congeners," Tetrahedron 49(9):1913-1924. cited by applicant
.
Toki et al., "Cures and regressions of established tumor xenographs
with monoclonal antibody," 223rd ACS Metting, Orlando FL, Apr.
7-11, 2002, Abstract 147. cited by applicant .
Tomioka et al., 1991, "An expeditious synthesis of dolastatin 10,"
Tetrahedron Letters 32(21):2395-2398. cited by applicant .
Trail et al., "Cure of Xenografted Human Carcinomas by
BR-96-Doxorubicin Immunoconjugates," Science, 1993, 261, 212-215.
cited by applicant .
Trail et al., "Effects of Linker Variation on the Stability,
Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin
Immunoconjugates," Cancer Research, 1997, 57, 100-105. cited by
applicant .
Tsutsumi et al., "Site-specific chemical modification with
polyethylene glycol of recombinant immunotoxin
anti-Tac(Fv)-PE38(LMB-2) improves Antitumor activity and reduces
animal toxicity and immunogenicity," PNAS, 2000, 97(15), 8548-8553.
cited by applicant .
Verdier-Pinard, P. et al., "Sustained Intracellular Retention of
Dolastatin 10 Causes Its Potent Antimitotic Activity," Molecular
Pharmacology, 2000, vol. 57, pp. 180-187. cited by applicant .
Wahl et al., "Anti-Cancer Activity of High Potency Anti-CD20
Antibody-Drug Conjugates," poster #769 at the annual meeting of the
American Association for Cancer Research, AACR, Toronto, Ontario,
CA, Apr. 5-9, 2003. cited by applicant .
Law et al., "CD70 is expressed on renal cell carcinoma and is a
potential target for turmor cell elimination by antibody-drug
conjugates," Proceedings of the AACR, 2004, vol. 45, abstract #625.
cited by applicant .
Pettit, 1997, "The Dolastatins," (reprint) Springer-Verlag, New
York 70:1-79. cited by applicant.
|
Primary Examiner: Jarrell; Noble
Attorney, Agent or Firm: Kilpatrick Townsend & Stockton
LLP
Parent Case Text
CONTINUITY
This application is a division of U.S. patent application Ser. No.
11/833,961, filed Aug. 3, 2007 which is a division of U.S.
application Ser. No. 10/983,340, filed Nov. 25, 2004, which claims
the benefit of U.S. Provisional Patent Application No. 60/622,455,
filed Oct. 27, 2004; U.S. Provisional Patent Application No.
60/598,899, filed Aug. 4, 2004; U.S. Provisional Patent Application
No. 60/557,116, filed Mar. 26, 2004; and U.S. Provisional Patent
Application No. 60/518,534, filed Nov. 6, 2003; the disclosures of
which are incorporated by reference herein.
Claims
What is claimed is:
1. An antibody-drug conjugate having the formula IIIa: ##STR00065##
or a pharmaceutically acceptable salt thereof, wherein: Ab is an
antibody that specifically binds to the CD19 antigen R.sup.17 is
selected from the group consisting of C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8-arylene-,
--C.sub.1-C.sub.10 alkylene-arylene-, -arylene-C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
carbocyclo)-, --(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10
alkylene-, --C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; r is an integer ranging
from 1-10; p ranges from 1 to about 20, and D is a drug moiety of
Formula D.sub.F: ##STR00066## wherein the wavy line of D.sub.F
indicates the covalent attachment site to C(O) in formula IIIa,
independently at each location: R.sup.2 is selected from the group
consisting of H and C.sub.1-C.sub.8 alkyl; R.sup.3 is selected from
the group consisting of H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); R.sup.4 is
selected from the group consisting of H, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl,
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8
heterocycle and C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8
heterocycle); R.sup.5 is selected from the group consisting of H
and methyl; or R.sup.4 and R.sup.5 jointly form a carbocyclic ring
and have the formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a
and R.sup.b are independently selected from the group consisting of
H, C.sub.1-C.sub.8 alkyl and C.sub.3-C.sub.8 carbocycle and n is
selected from the group consisting of 2, 3, 4, 5 and 6; R.sup.6 is
selected from the group consisting of H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from the group consisting of H, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle, aryl, C.sub.1-C.sub.8
alkyl-aryl, C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle); each R.sup.8 is independently
selected from the group consisting of H, OH, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8 alkyl); R.sup.9
is selected from the group consisting of H and C.sub.1-C.sub.8
alkyl; R.sup.10 is selected from aryl or C.sub.3-C.sub.8
heterocycle; Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl; R.sup.11 is selected from H,
C.sub.1-C.sub.20 alkyl, aryl, C.sub.3-C.sub.8 heterocycle,
--(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2; m is an integer ranging
from 1-1000; R.sup.13 is C.sub.2-C.sub.8 alkyl; R.sup.14 is H or
C.sub.1-C.sub.8 alkyl; each occurrence of R.sup.15 is independently
H, COOH, --(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl; each
occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8 alkyl,
or --(CH.sub.2).sub.n--COOH; and n is an integer ranging from 0 to
6.
2. The antibody-drug conjugate of claim 1, wherein the antibody is
attached to the drug moiety through a cysteine residue of the
antibody.
3. The antibody-drug conjugate of claim 2, wherein p is 2 to 5.
4. The antibody-drug conjugate of claim 1, wherein p is 2 to 8.
5. The antibody-drug conjugate of claim 1, wherein p is 2 to 5.
6. The antibody-drug conjugate of claim 1, having the formula:
##STR00067## or a pharmaceutically acceptable salt thereof.
7. The antibody-drug conjugate of claim 6, wherein p is 2 to 4 and
D has the formula: ##STR00068## or a pharmaceutically acceptable
salt thereof.
8. The antibody-drug conjugate of claim 1, wherein D has the
formula: ##STR00069## or a pharmaceutically acceptable salt
thereof.
9. The antibody-drug conjugate of claim 1, wherein D has the
formula: ##STR00070## or a pharmaceutically acceptable salt
thereof.
10. The antibody-drug conjugate of claim 1, wherein D has the
formula: ##STR00071## or a pharmaceutically acceptable salt
thereof.
11. The antibody-drug conjugate of claim 1, wherein D has the
formula: ##STR00072## or a pharmaceutically acceptable salt
thereof.
12. The antibody-drug conjugate of claim 1, wherein D has the
formula: ##STR00073## or a pharmaceutically acceptable salt
thereof.
13. The antibody-drug conjugate of claim 1, wherein the antibody is
a monoclonal antibody.
14. An antibody-drug conjugate of claim 1, having the formula:
##STR00074## or a pharmaceutically acceptable salt thereof.
15. A pharmaceutical composition comprising an effective amount of
the antibody-drug conjugate of claim 1 and a pharmaceutically
acceptable diluent, carrier or excipient.
Description
JOINT RESEARCH AGREEMENT
Some of the subject matter in this application was made by or on
behalf of Seattle Genetics, Inc. and Genentech, Inc. as a result of
activities undertaken within the scope of a joint research
agreement effective on or before the date the claimed invention was
made.
1. FIELD OF THE INVENTION
The present invention is directed to a Drug Compound and more
particularly to Drug-Linker-Ligand Conjugates, Drug-Linker
Compounds, and Drug-Ligand Conjugates, to compositions including
the same, and to methods for using the same to treat cancer, an
autoimmune disease or an infectious disease. The present invention
is also directed to antibody-drug conjugates, to compositions
including the same, and to methods for using the same to treat
cancer, an autoimmune disease or an infectious. disease. The
invention also relates to methods of using antibody-drug conjugate
compounds for in vitro, in situ, and in vivo diagnosis or treatment
of mammalian cells, or associated pathological conditions.
2. BACKGROUND OF THE INVENTION
Improving the delivery of drugs and other agents to target cells,
tissues and tumors to achieve maximal efficacy and minimal toxicity
has been the focus. of considerable research for many years. Though
many attempts have been made to develop effective methods for
importing biologically active molecules into cells, both in vivo
and in vitro, none has proved to be entirely satisfactory.
Optimizing the association of the drug with its intracellular
target, while minimizing intercellular redistribution of the drug,
e.g., to neighboring cells, is often difficult or inefficient.
Most agents currently administered to a patient parenterally are
not targeted, resulting in systemic delivery of the agent to cells
and tissues of the body where it is unnecessary, and often
undesirable. This may result in adverse drug side effects, and
often limits the dose of a drug (e.g., chemotherapeutic
(anti-cancer), cytotoxic, enzyme inhibitor agents and antiviral or
antimicrobial drugs) that can be administered. By comparison,
although oral administration of drugs is considered to be a
convenient and economical mode of administration, it shares the
same concerns of non-specific toxicity to unaffected cells once the
drug has been absorbed into the systemic circulation. Further
complications involve problems with oral bioavailability and
residence of drug in the gut leading to additional exposure of gut
to the drug and hence risk of gut toxicities. Accordingly, a major
goal has been to develop methods for specifically targeting agents
to cells and tissues. The benefits of such treatment include
avoiding the general physiological effects of inappropriate
delivery of such agents to other cells and tissues, such as
uninfected cells. Intracellular targeting may be achieved by
methods, compounds and formulations which allow accumulation or
retention of biologically active agents, i.e. active metabolites,
inside cells.
Monoclonal antibody therapy has been established for the targeted
treatment of patients with cancer, immunological and angiogenic
disorders.
The use of antibody-drug conjugates for the local delivery of
cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg. Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278)
theoretically allows targeted delivery of the drug moiety to
tumors, and intracellular accumulation therein, while systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet
pp. (Mar. 15, 1986):603-05; Thorpe, 1985, "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., 1986, supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Kerr et al., 1997, Bioconjugate Chem.
8(6):781-784; Mandler et al. (2000) Jour. of the Nat. Cancer Inst.
92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem.
Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al.
(1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res.
53:3336-3342). The toxins may affect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition (Meyer, D. L. and Senter, P. D. "Recent
Advances in Antibody Drug Conjugates for Cancer Therapy" in Annual
Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237).
Some cytotoxic drugs tend to be inactive or less active when
conjugated to large antibodies or protein receptor ligands.
ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al.
(2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate
composed of a hu CD33 antibody linked to calicheamicin, was
approved in 2000 for the treatment of acute myeloid leukemia by
injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;
5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an
antibody drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an
antibody drug conjugate composed of the anti-prostate specific
membrane antigen (PSMA) monoclonal antibody linked to the
maytansinoid drug moiety, DM1, is under development for the
potential treatment of prostate tumors. The same maytansinoid drug
moiety, DM1, was linked through a non-disulfide linker, SMCC, to a
mouse murine monoclonal antibody, TA.1 (Chari et al. (1992) Cancer
Research 52:127-131). This conjugate was reported to be 200-fold
less potent than the corresponding disulfide linker conjugate. The
SMCC linker was considered therein to be "noncleavable."
Several short peptidic compounds have been isolated from the marine
mollusc Dolabella auricularia and found to have biological activity
(Pettit et al. (1993) Tetrahedron 49:9151; Nakamura et al. (1995)
Tetrahedron Letters 36:5059-5062; Sone et al. (1995) Jour. Org
Chem. 60:4474). Analogs of these compounds have also been prepared,
and some were found to have biological activity (for a review, see
Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277). For
example, auristatin E (U.S. Pat. No. 5,635,483) is a synthetic
analogue of the marine natural product Dolastatin 10, an agent that
inhibits tubulin polymerization by binding to the same domain on
tubulin as the anticancer drug vincristine (G. R. Pettit, (1997)
Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin 10, auristatin PE,
and auristatin E are linear peptides having four amino acids, three
of which are unique to the dolastatin class of compounds, and a
C-terminal amide.
The auristatin peptides, auristain E (AE) and monomethylauristatin
(MMAE), synthetic analogs of dolastatin, were conjugated to: (i)
chimeric monoclonal antibodies cBR96 (specific to Lewis Y on
carcinomas); (ii) cAC10 which is specific to CD30 on hematological
malignancies (Klussman, et al. (2004), Bioconjugate Chemistry
15(4):765-773; Doronina et al. (2003) Nature Biotechnology
21(7):778-784; "Monomethylvaline Compounds Capable of Conjugation
to Ligands"; Francisco et al. (2003) Blood 102(4):1458-1465; U.S.
Publication 2004/0018194; (iii) anti-CD20 antibodies such as
RITUXAN.RTM. (WO 04/032828) for the treatment of CD20-expressing
cancers and immune disorders; (iv) anti-EphB2 antibodies 2H9 and
anti-IL-8 for treatment of colorectal cancer (Mao, et al. (2004)
Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskar et
al. (2003) Cancer Res. 63:6387-6394); and (vi) other anti-CD30
antibodies (WO 03/043583).
Auristatin E conjugated to monoclonal antibodies are disclosed in
Senter et al, Proceedings of the American Association for Cancer
Research, Volume 45, Abstract Number 623, presented Mar. 28,
2004.
Despite in vitro data for compounds of the dolastatin class and its
analogs, significant general toxicities at doses required for
achieving a therapeutic effect compromise their efficacy in
clinical studies. Accordingly, there is a clear need in the art for
dolastatin/auristatin derivatives having significantly lower
toxicity, yet useful therapeutic efficiency. These and other
limitations and problems of the past are addressed by the present
invention.
The ErbB family of receptor tyrosine kinases are important
mediators of cell growth, differentiation and survival. The
receptor family includes four distinct members including epidermal
growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of
anti-ErbB2 antibodies has been characterized using the human breast
tumor cell line SKBR3 (Hudziak et al., (1989) Mol. Cell. Biol.
9(3):1165-1172. Maximum inhibition was obtained with the antibody
called 4D5 which inhibited cellular proliferation by 56%. Other
antibodies in the panel reduced cellular proliferation to a lesser
extent in this assay. The antibody 4D5 was further found to
sensitize ErbB2-overexpressing breast tumor cell lines to the
cytotoxic effects of TNF-.alpha. (U.S. Pat. No. 5,677,171). The
anti-ErbB2 antibodies discussed in Hudziak et al. are further
characterized in Fendly et al. (1990) Cancer Research 50:1550-1558;
Kotts et al. (1990) In vitro 26(3):59A; Sarup et al. (1991) Growth
Regulation 1:72-82; Shepard et al. J. (1991) Clin. Immunol.
11(3):117-127; Kumar et al. (1991) Mol. Cell. Biol. 11(2):979-986;
Lewis et al. (1993) Cancer Immunol. Immunother. 37:255-263; Pietras
et al. (1994) Oncogene 9:1829-1838; Vitetta et al. (1994) Cancer
Research 54:5301-5309; Sliwkowski et al. (1994) J. Biol. Chem.
269(20):14661-14665; Scott et al. (1991).sub.3. Biol. Chem.
266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994)
91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465; and
Schaefer et al. (1997) Oncogene 15:1385-1394.
Other anti-ErbB2 antibodies with various properties have been
described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991);
McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res.
51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis
3:350-362 (1990); Stancovski et al. Proc. Natl. Acad. Sci. USA
88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589
(1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94/00136;
Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al.
(1991) Cancer Res. 51:4575-4580; Shawver et al. (1994) Cancer Res.
54:1367-1373; Arteaga et al. (1994) Cancer Res. 54:3758-3765;
Harwerth et al. (1992) J. Biol. Chem. 267:15160-15167; U.S. Pat.
No. 5,783,186; and Klapper et al. (1997) Oncogene 14:2099-2109.
Homology screening has resulted in the identification of two other
ErbB receptor family members; ErbB3 (U.S. Pat. No. 5,183,884; U.S.
Pat. No. 5,480,968; KraU. S. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993) Proc.
Natl. Acad. Sci. USA 90:1746-1750; and Plowman et al. (1993) Nature
366:473-475). Both of these receptors display increased expression
on at least some breast cancer cell lines.
HERCEPTIN.RTM. (Trastuzumab) is a recombinant DNA-derived humanized
monoclonal antibody that selectively binds with high affinity in a
cell-based assay (Kd=5 nM) to the extracellular domain of the human
epidermal growth factor receptor2 protein, HER2 (ErbB2) (U.S. Pat.
No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213;
U.S. Pat. No. 6,639,055; Coussens L, et al. (1985) Science
230:1132-9; Slamon D J, et al. (1989) Science 244:707-12).
Trastuzumab is an IgG1 kappa antibody that contains human framework
regions with the complementarity-determining regions of a murine
antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2
antigen and thus. inhibits the growth of cancerous cells. Because
Trastuzumab is a humanized antibody, it minimizes any HAMA response
in patients. The humanized antibody against HER2 is produced by a
mammalian cell (Chinese Hamster Ovary, CHO) suspension culture. The
HER2 (or c-erbB2) proto-oncogene encodes a transmembrane receptor
protein of 185 kDa, which is structurally related to the epidermal
growth factor receptor. HER2 protein overexpression is observed in
25%-30% of primary breast cancers and can be determined using an
immunohistochemistry based assessment of fixed tumor blocks (Press
M F, et al. (1993) Cancer Res 53:4960-70. Trastuzumab has been
shown, in both in vitro assays and in animals, to inhibit the
proliferation of human tumor cells that overexpress HER2 (Hudziak R
M, et al. (1989) Mol Cell Biol 9:1165-72; Lewis G D, et al. (1993)
Cancer Immunol Immunother; 37:255-63; Baselga J, et al. (1998)
Cancer Res. 58:2825-2831). Trastuzumab is a mediator of
antibody-dependent cellular cytotoxicity, ADCC (Hotaling T E, et
al. (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res;
37:471; Pegram M D, et al. (1997) [abstract]. Proc Am Assoc Cancer
Res; 38:602). In vitro, Trastuzumab mediated ADCC has been shown to
be preferentially exerted on HER2 overexpressing cancer cells
compared with cancer cells that do not overexpress HER2.
HERCEPTIN.RTM. as a single agent is indicated for the treatment of
patients with metastatic breast cancer whose tumors overexpress the
HER2 protein and who have received one or more chemotherapy
regimens for their metastatic disease. HERCEPTIN.RTM. in
combination with paclitaxel is indicated for treatment of patients
with metastatic breast cancer whose tumors overexpress the HER2
protein and who have not received chemotherapy for their metastatic
disease. HERCEPTIN.RTM. is clinically active in patients with
ErbB2-overexpressing metastatic breast cancers that have received
extensive prior anti-cancer therapy (Baselga et al, (1996) J. Clin.
Oncol. 14:737-744).
The murine monoclonal anti-HER2 antibody inhibits the growth of
breast cancer cell lines that overexpress HER2 at the 2+ and 3+
(1-2.times.10.sup.6 HER2 receptors per cell) level, but has no
activity on cells that express lower levels of HER2 (Lewis et al.,
(1993) Cancer Immunol. Immunother. 37:255-263). Based on this
observation, antibody 4D5 was humanized (huMAb4D5-8, rhuMAb HER2,
U.S. Pat. No. 5,821,337; Carter et al., (1992) Proc. Natl. Acad.
Sci. USA 89: 4285-4289) and tested in breast cancer patients whose
tumors overexpress HER2 but who had progressed after conventional
chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17:
2639-2648).
Although HERCEPTIN is a breakthrough in treating patients with
ErbB2-overexpressing breast cancers that have received extensive
prior anti-cancer therapy, some patients in this population fail to
respond or respond only poorly to HERCEPTIN treatment.
Therefore, there is a significant clinical need for developing
further HER2-directed cancer therapies for those patients with
HER2-overexpressing tumors or other diseases associated with HER2
expression that do not respond, or respond poorly, to HERCEPTIN
treatment.
The recitation of any reference in this application is not an
admission that the reference is prior art to this application.
3. SUMMARY OF THE INVENTION
In one aspect, the present invention provides Drug-Linker-Ligand
compounds having the Formula Ia: LA.sub.a-W.sub.w--Y.sub.y-D).sub.p
Ia
or a pharmaceutically acceptable salt or solvate thereof
wherein,
L- is a Ligand unit;
-A.sub.a-W.sub.w--Y.sub.y-- is a Linker unit (LU), wherein the
Linker unit includes:
-A- is a Stretcher unit,
a is 0 or 1,
each --W-- is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
--Y-- is a Spacer unit, and
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the Formulas D.sub.E and D.sub.F:
##STR00001##
wherein, independently at each location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH; where; n is an integer ranging
from 0 to 6; and
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
carbocycle).
In another aspect, Drug Compounds having the Formula Ib are
provided:
##STR00002## or pharmaceutically acceptable salts or solvates
thereof,
wherein:
R.sup.2 is selected from hydrogen and --C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 jointly, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are
attached;
R.sup.6 is selected from H and --C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, --OH,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl);
R.sup.9 is selected from H and --C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl group or --C.sub.3-C.sub.8
heterocycle;
Z is --O--, --S--, --NH--, or --NR.sup.12--, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
--C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.n--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is --C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or --C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, --C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH; and
n is an integer ranging from 0 to 6.
The compounds of Formula (Ib) are useful for treating cancer, an
autoimmune disease or an infectious disease in a patient or useful
as an intermediate for the synthesis of a Drug-Linker,
Drug-Linker-Ligand Conjugate, and Drug-Ligand Conjugate having a
cleavable Drug unit.
In another aspect, compositions are provided including an effective
amount of a Drug-Linker-Ligand Conjugate and a pharmaceutically
acceptable carrier or vehicle.
In still another aspect, the invention provides pharmaceutical
compositions comprising an effective amount of a Drug-Linker
Compound and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the invention provides compositions
comprising an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate and a
pharmaceutically acceptable carrier or vehicle.
In yet another aspect, the invention provides methods for killing
or inhibiting the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or
inhibiting the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for killing or
inhibiting the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Ligand Conjugate having a cleavable Drug unit from
the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for
treating cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating
cancer including administering to a patient in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating
cancer including administering to a patient in need thereof an
effective amount of a Drug-Ligand Conjugate having a cleavable Drug
unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for killing
or inhibiting the replication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or
inhibiting the replication of a cell that expresses an autoimmune
antibody including administering to a patient in need thereof an
effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for killing or
inhibiting the replication of a cell that expresses an autoimmune
antibody including administering to a patient in need thereof an
effective amount of a Drug-Ligand Conjugate having a cleavable Drug
unit from the Drug-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating
an autoimmune disease including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating
an autoimmune disease including administering to a patient in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating
an autoimmune disease including administering to a patient in need
thereof an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for
treating an infectious. disease including administering to a
patient in need thereof an effective amount of a Drug-Linker
Compound.
In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Ligand Conjugate
having a cleavable Drug unit from the Drug-Ligand Conjugate.
In yet another aspect, the invention provides methods for
preventing the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing
the multiplication of a tumor cell or cancer cell including
administering to a patient in need thereof an effective amount of a
Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for preventing
the multiplication of a tumor cell or cancer cell including
administering to a patient in need thereof an effective amount of a
Drug-Ligand Conjugate having a cleavable Drug unit from the
Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for
preventing the multiplication of a cell that expresses an
autoimmune antibody including administering to a patient in need
thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing
the multiplication of a cell that expresses an autoimmune antibody
including administering to a patient in need thereof an effective
amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for preventing
the multiplication of a cell that expresses an autoimmune antibody
including administering to a patient in need thereof an effective
amount of a Drug-Ligand Conjugate having a cleavable Drug unit from
the Drug-Ligand Conjugate.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Linker
Compound.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Ligand
Conjugate having a cleavable Drug unit from the Drug-Ligand
Conjugate.
In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Linker
Compound.
In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Ligand
Conjugate having a cleavable Drug unit from the Drug-Ligand
Conjugate.
In another aspect, a Drug Compound is provided which can be used as
an intermediate for the synthesis of a Drug-Linker Compound having
a cleavable Drug unit from the Drug-Ligand Conjugate.
In another aspect, a Drug-Linker Compound is provided which can be
used as an intermediate for the synthesis of a Drug-Linker-Ligand
Conjugate.
In another aspect, compounds having Formula Ia' are provided:
AbA.sub.a-W.sub.w--Y.sub.y-D).sub.p Ia'
or a pharmaceutically acceptable salt or solvate thereof, wherein:
Ab includes an antibody including one which binds to CD30, CD40,
CD70, and Lewis Y antigen,
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a Drug unit selected from Formulas D.sub.E and D.sub.F:
##STR00003##
wherein, independently at each location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH;
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
n is an integer ranging from 0 to 6.
In one embodiment, Ab is not an antibody which binds to an ErbB
receptor or which binds to one or more of receptors (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank
accession no. NM.sub.--001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.--003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate,
Genbank accession no. NM.sub.--012449);
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
(7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
(11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no.
AF455138);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor
potential cation channel, subfamily M, member 4, Genbank accession
no. NM.sub.--017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived
growth factor, Genbank accession no. NP.sub.--003203 or
NM.sub.--003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr
virus receptor) or Hs.73792, Genbank accession no. M26004);
(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank
accession no. NM.sub.--000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase
anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM
030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20R.alpha. (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM.sub.--004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763);
(26) BAFF-R (Genbank accession no. NP.sub.--443177.1);
(27) CD22 (Genbank accession no. NP-001762.1);
(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation, Genbank
accession No. NP.sub.--001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled
receptor that is activated by the CXCL13 chemokine, functions in
lymphocyte migration and humoral defense, plays a role in HIV-2
infection and perhaps development of AIDS, lymphoma, myeloma, and
leukemia, Genbank accession No. NP.sub.--001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)
that binds peptides and presents them to CD4+ T lymphocytes,
Genbank accession No. NP.sub.--002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an
ion channel gated by extracellular ATP, may be involved in synaptic
transmission and neurogenesis, deficiency may contribute to the
pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank
accession No. NP.sub.--001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the
immunoglobulin Fc domain that contains C2 type Ig-like and ITAM
domains, may have a role in B-lymphocyte differentiation, Genbank
accession No. NP.sub.--443170.1); or
(35) IRTA2 (Immunoglobulin superfamily receptor translocation
associated 2, a putative immunoreceptor with possible roles in B
cell development and lymphomagenesis; deregulation of the gene by
translocation occurs in some B cell malignancies, Genbank accession
No. NP.sub.--112571.1).
In still another aspect, the invention provides pharmaceutical
compositions comprising an effective amount of a
Drug-Linker-Antibody Conjugate and a pharmaceutically acceptable
carrier or vehicle.
In still another aspect, the invention provides compositions
comprising an effective amount of a Drug-Antibody Conjugate having
a cleavable Drug unit (moiety) from the Drug-Antibody Conjugate and
a pharmaceutically acceptable carrier or vehicle.
In another aspect, the invention provides methods for killing or
inhibiting the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for killing or
inhibiting the multiplication of a tumor cell or cancer cell
including administering to a patient in need thereof an effective
amount of a Drug-Antibody Conjugate having a cleavable Drug unit
from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating
cancer including administering to a patient in need thereof an
effective amount of a Drug-Linker-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating
cancer including administering to a patient in need thereof an
effective amount of a Drug-Antibody Conjugate having a cleavable
Drug unit from the Drug-Antibody Conjugate.
In another aspect, the invention provides methods for killing or
inhibiting the replication of a cell that expresses an autoimmune
antibody including administering to a patient in need thereof an
effective amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for killing or
inhibiting the replication of a cell that expresses an autoimmune
antibody including administering to a patient in need thereof an
effective amount of a Drug-Antibody Conjugate having a cleavable
Drug unit from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating
an autoimmune disease including administering to a patient in need
thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
In yet another aspect, the invention provides methods for treating
an autoimmune disease including administering to a patient in need
thereof an effective amount of a Drug-Antibody Conjugate having a
cleavable Drug unit from the Drug-Antibody Conjugate.
In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
In still another aspect, the invention provides methods for
treating an infectious disease including administering to a patient
in need thereof an effective amount of a Drug-Antibody Conjugate
having a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, the invention provides methods for preventing
the multiplication of a tumor cell or cancer cell including
administering to a patient in need thereof an effective amount of a
Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for preventing
the multiplication of a tumor cell or cancer cell including
administering to a patient in need thereof an effective amount of a
Drug-Antibody Conjugate having a cleavable Drug unit from the
Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Linker-Antibody
Conjugate.
In yet another aspect, the invention provides methods for
preventing cancer including administering to a patient in need
thereof an effective amount of a Drug-Antibody Conjugate having a
cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, the invention provides methods for preventing
the multiplication of a cell that expresses an autoimmune antibody
including administering to a patient in need thereof an effective
amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for preventing
the multiplication of a cell that expresses an autoimmune antibody
including administering to a patient in need thereof an effective
amount of a Drug-Antibody Conjugate having a cleavable Drug unit
from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a
Drug-Linker-Antibody Conjugate.
In yet another aspect, the invention provides methods for
preventing an autoimmune disease including administering to a
patient in need thereof an effective amount of a Drug-Antibody
Conjugate having a cleavable Drug unit from the Drug-Antibody
Conjugate.
In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a
Drug-Linker-Antibody Conjugate.
In still another aspect, the invention provides methods for
preventing an infectious disease including administering to a
patient in need thereof an effective amount of a Drug-Antibody
Conjugate having a cleavable Drug unit from the Drug-Antibody
Conjugate.
In another aspect, a Drug Compound is provided which can be used as
an intermediate for the synthesis of a Drug-Linker Compound having
a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, a Drug-Linker Compound is provided which can be
used as an intermediate for the synthesis of a Drug-Linker-Antibody
Conjugate.
In one aspect, the present invention provides Drug-Linker-Antibody
Conjugates (also referred to as antibody-drug conjugates) having
Formula Ic: AbA.sub.a-W.sub.w--Y.sub.y-D).sub.p Ic
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
Ab is an antibody which binds to one or more of the antigens
(1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank
accession no. NM.sub.--001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.--003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate,
Genbank accession no. NM.sub.--012449);
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
(7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
(11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no.
AF455138);
(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor
potential cation channel, subfamily M, member 4, Genbank accession
no. NM.sub.--017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGFI, teratocarcinoma-derived
growth factor, Genbank accession no. NP.sub.--003203 or
NM.sub.--003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr
virus receptor) or Hs.73792, Genbank accession no. M26004);
(15) CD79b (1 Gb (immunoglobulin-associated beta), B29, Genbank
accession no. NM.sub.--000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase
anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no.
NM.sub.--030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20R.alpha. (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM.sub.--004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763);
(26) BAFF-R (Genbank accession no. NP.sub.--443177.1);
(27) CD22 (Genbank accession no. NP-001762.1);
(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation, Genbank
accession No. NP.sub.--001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled
receptor that is activated by the CXCL13 chemokine, functions in
lymphocyte migration and humoral defense, plays a role in HIV-2
infection and perhaps development of AIDS, lymphoma, myeloma, and
leukemia, Genbank accession No. NP.sub.--001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)
that binds peptides and presents them to CD4+ T lymphocytes,
Genbank accession No. NP.sub.--002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an
ion channel gated by extracellular ATP, may be involved in synaptic
transmission and neurogenesis, deficiency may contribute to the
pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank
accession No. NP.sub.--001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the
immunoglobulin Fc domain that contains C2 type Ig-like and ITAM
domains, may have a role in B-lymphocyte differentiation, Genbank
accession No. NP.sub.--443170.1); or
(35) IRTA2 (Immunoglobulin superfamily receptor translocation
associated 2, a putative immunoreceptor with possible roles in B
cell development and lymphomagenesis; deregulation of the gene by
translocation occurs in some B cell malignancies, Genbank accession
No. NP.sub.--112571.1);
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a Drug moiety selected from Formulas D.sub.E and D.sub.F:
##STR00004##
wherein the wavy line of D.sub.E and D.sub.F indicates the covalent
attachment site to A, W, or Y, and independently at each
location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O)m-R.sup.14, or
--(R.sup.13O)m-CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2)n-N(R.sup.16).sub.2, --(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2)n-SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH;
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sup.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
n is an integer ranging from 0 to 6.
In another aspect, the antibody of the antibody-drug conjugate
(ADC) of the invention specifically binds to a receptor encoded by
an ErbB2 gene.
In another aspect, the antibody of the antibody-drug conjugate is a
humanized antibody selected from huMAb4D5-1, huMAb4D5-2,
huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and
huMAb4D5-8 (Trastuzumab).
In another aspect, the invention includes an article of manufacture
comprising an antibody-drug conjugate compound of the invention; a
container; and a package insert or label indicating that the
compound can be used to treat cancer characterized by the
overexpression of an ErbB2 receptor.
In another aspect, the invention includes a method for the
treatment of cancer in a mammal, wherein the cancer is
characterized by the overexpression of an ErbB2 receptor and does
not respond, or responds poorly, to treatment with an anti-ErbB2
antibody, comprising administering to the mammal a therapeutically
effective amount of an antibody-drug conjugate compound of the
invention.
In another aspect, a substantial amount of the drug moiety is not
cleaved from the antibody until the antibody-drug conjugate
compound enters a cell with a cell-surface receptor specific for
the antibody of the antibody-drug conjugate, and the drug moiety is
cleaved from the antibody when the antibody-drug conjugate does
enter the cell.
In another aspect, the bioavailability of the antibody-drug
conjugate compound or an intracellular metabolite of the compound
in a mammal is improved when compared to a drug compound comprising
the drug moiety of the antibody-drug conjugate compound, or when
compared to an analog of the compound not having the drug
moiety.
In another aspect, the drug moiety is intracellularly cleaved in a
mammal from the antibody of the compound, or an intracellular
metabolite of the compound.
In another aspect, the invention includes a pharmaceutical
composition comprising an effective amount of the antibody-drug
conjugate compound of the invention, or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable diluent,
carrier or excipient. The composition may further comprise a
therapeutically effective amount of chemotherapeutic agent such as
a tubulin-forming inhibitor, a topoisomerase inhibitor, and a DNA
binder.
In another aspect, the invention includes a method for killing or
inhibiting the proliferation of tumor cells or cancer cells
comprising treating tumor cells or cancer cells with an amount of
the antibody-drug conjugate compound of the invention, or a
pharmaceutically acceptable salt or solvate thereof, being
effective to kill or inhibit the proliferation of the tumor cells
or cancer cells.
In another aspect, the invention includes a method of inhibiting
cellular proliferation comprising exposing mammalian cells in a
cell culture medium to an antibody drug conjugate compound of the
invention, wherein the antibody drug conjugate compound enters the
cells and the drug is cleaved from the remainder of the antibody
drug conjugate compound; whereby proliferation of the cells is
inhibited.
In another aspect, the invention includes a method of treating
cancer comprising administering to a patient a formulation of an
antibody-drug conjugate compound of the invention and a
pharmaceutically acceptable diluent, carrier or excipient.
In another aspect, the invention includes an assay for detecting
cancer cells comprising: (a) exposing cells to an antibody-drug
conjugate compound of the invention; and (b) determining the extent
of binding of the antibody-drug conjugate compound to the
cells.
The invention will best be understood by reference to the following
detailed description of the exemplary embodiments, taken in
conjunction with the accompanying drawings, figures, and schemes.
The discussion below is descriptive, illustrative and exemplary and
is not to be taken as limiting the scope defined by any appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an in vivo, single dose, efficacy assay of
cAC10-mcMMAF in subcutaneous Karpas-299 ALCL xenografts.
FIG. 2 shows an in vivo, single dose, efficacy assay of
cAC10-mcMMAF in subcutaneous L540cy. For this study there were 4
mice in the untreated group and 10 in each of the treatment
groups.
FIGS. 3a and 3b show in vivo efficacy of cBR96-mcMMAF in
subcutaneous L2987. The filed triangles in FIG. 3a and arrows in
FIG. 3b indicate the days of therapy.
FIGS. 4a and 4b show in vitro activity of cAC10-antibody-drug
conjugates against CD30.sup.+ cell lines.
FIGS. 5a and 5b show in vitro activity of cBR96-antibody-drug
conjugates against LeY.sup.+ cell lines.
FIGS. 6a and 6b show in vitro activity of c1F6-antibody-drug
conjugates against CD70.sup.+ renal cell carcinoma cell lines.
FIG. 7 shows an in vitro, cell proliferation assay with SK-BR-3
cells treated with antibody drug conjugates (ADC):
-.circle-solid.-Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab,
-.smallcircle.-Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and
-.DELTA.-Trastuzumab-MC-MMAF, 4.8 MMAF/Ab, measured in Relative
Fluorescence Units (RLU) versus .mu.g/ml concentration of ADC.
H=Trastuzumab where H is linked via a cysteine [cys].
FIG. 8 shows an in vitro, cell proliferation assay with BT-474
cells treated with ADC: -.circle-solid.-Trastuzumab-MC-vc-PAB-MMAF,
3.8 MMAF/Ab, -.smallcircle.-Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and
-.DELTA.-Trastuzumab-MC-MMAF, 4.8 MMAF/Ab.
FIG. 9 shows an in vitro, cell proliferation assay with MCF-7 cells
treated with ADC: -.circle-solid.-Trastuzumab-MC-vc-PAB-MMAF, 3.8
MMAF/Ab, -.smallcircle.-Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9
MMAF/Ab, and -.DELTA.-Trastuzumab-MC-MMAF, 4.1 MMAF/Ab.
FIG. 10 shows an in vitro, cell proliferation assay with MDA-MB-468
cells treated with ADC: -.circle-solid.-Trastuzumab-MC-vc-PAB-MMAE,
4.1 MMAE/Ab, -.smallcircle.-Trastuzumab-MC-vc-PAB-MMAE, 3.3
MMAE/Ab, and -.DELTA.-Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.
FIG. 11 shows a plasma concentration clearance study after
administration of H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to
Sprague-Dawley rats: The administered dose was 2 mg of ADC per kg
of rat. Concentrations of total antibody and ADC were measured over
time. (H=Trastuzumab).
FIG. 12 shows a plasma concentration clearance study after
administration of H-MC-vc-MMAE to Cynomolgus monkeys at different
doses: 0.5, 1.5, 2.5, and 3.0 mg/kg administered at day 1 and day
21. Concentrations of total antibody and ADC were measured over
time. (H=Trastuzumab).
FIG. 13 shows the mean tumor volume change over time in athymic
nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day
0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 .mu.g/m.sup.2)
and Trastuzumab-MC-vc-PAB-MMAF (555 .mu.g/m.sup.2).
(H=Trastuzumab).
FIG. 14 shows the mean tumor volume change over time in athymic
nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day
0 with 10 mg/kg (660 .mu.g/m.sup.2) of Trastuzumab-MC-MMAE and 1250
.mu.g/m.sup.2 Trastuzumab-MC-vc-PAB-MMAE.
FIG. 15 shows the mean tumor volume change over time in athymic
nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day
0 with Vehicle and 650 .mu.g/m.sup.2 trastuzumab-MC-MMAF.
FIG. 16 shows the mean tumor volume change over time in athymic
nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day
0 with Vehicle and 350 .mu.g/m.sup.2 of four trastuzumab-MC-MMAF
conjugates where the MMAF/trastuzumab (H) ratio is 2, 4, 5.9 and
6.
FIG. 17 shows the Group mean change, with error bars, in animal
(rat) body weights (Mean.+-.SD) after administration of Vehicle,
trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF,
trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF.
FIG. 18 shows the Group mean change in animal (rat) body weights
(Mean.+-.SD) after administration of 9.94 mg/kg H-MC-vc-MMAF, 24.90
mg/kg H-MC-vc-MMAF, 10.69 mg/kg H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kg
H-MC(Me)-vc-PAB-MMAF, 10.17 mg/kg H-MC-MMAF, 25.50 mg/kg H-MC-MMAF,
and 21.85 mg/kg H-MC-vc-PAB-MMAF. H=trastuzumab. The MC linker is
attached via a cysteine of trastuzumab for each conjugate.
FIG. 19 shows the Group mean change, with error bars, in Sprague
Dawley rat body weights (Mean.+-.SD) after administration of
trastuzumab (H)-MC-MMAF at doses of 2105, 3158, and 4210
.mu.g/m.sup.2. The MC linker is attached via a cysteine of
trastuzumab for each conjugate.
FIG. 20 shows examples of compounds with a non self-immolative
Spacer unit.
FIG. 21 shows a scheme of a possible mechanism of Drug release from
a PAB group which is attached directly to -D via a carbamate or
carbonate group.
FIG. 22 shows a scheme of a possible mechanism of Drug release from
a PAB group which is attached directly to -D via an ether or amine
linkage.
FIG. 23 shows an example of a branched spacer unit,
bis(hydroxymethyl)styrene (BHMS) unit, which can be used to
incorporate and release multiple drug.
FIG. 24 shows a scheme of the CellTiter-Glo.RTM. Assay.
FIG. 25 shows the synthesis of an N-terminal tripeptide unit F
which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
FIG. 26 shows the synthesis of an N-terminal tripeptide unit F
which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
FIG. 27 shows the synthesis of an N-terminal tripeptide unit F
which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
FIG. 28 shows the synthesis of useful linkers.
FIG. 29 shows the synthesis of useful linkers.
FIG. 30 shows a general synthesis of an illustrative Linker unit
containing a maleimide Stretcher group and optionally a
p-aminobenzyl ether self-immolative Spacer.
FIG. 31 shows the synthesis of a val-cit dipeptide Linker having a
maleimide Stretcher and optionally a p-aminobenzyl self-immolative
Spacer.
FIG. 32 shows the synthesis of a phe-lys(Mtr) dipeptide Linker unit
having a maleimide Stretcher unit and a p-aminobenzyl
self-immolative Spacer unit.
FIG. 33 shows the synthesis of a Drug-Linker Compound that contains
an amide or carbamate group, linking the Drug unit to the Linker
unit.
FIG. 34 shows illustrative methods useful for linking a Drug to a
Ligand to form a Drug-Linker Compound.
FIG. 35 shows the synthesis of a val-cit dipeptide linker having a
maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS)
unit.
FIG. 36 shows methodology useful for making Drug-Linker-Ligand
conjugates having about 2 to about 4 drugs per antibody.
FIG. 37 shows the synthesis of MC-MMAF via t-butyl ester.
FIG. 38 shows the synthesis of MC-MMAF (11) via dimethoxybenzyl
ester.
FIG. 39 shows the synthesis of analog of mc-MMAF.
4. DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
4.1 Definitions and Abbreviations
Unless stated otherwise, the following terms and phrases as used
herein are intended to have the following meanings:
When trade names are used herein, applicants intend to
independently include the trade name product formulation, the
generic drug, and the active pharmaceutical ingredient(s) of the
trade name product.
The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments,
so long as they exhibit the desired biological activity. An
antibody is a protein generated by the immune system that is
capable of recognizing and binding to a specific antigen. Described
in terms of its structure, an antibody typically has a Y-shaped
protein consisting of four amino acid chains, two heavy and two
light. Each antibody has primarily two regions: a variable region
and a constant region. The variable region, located on the ends of
the arms of the Y, binds to and interacts with the target antigen.
This variable region includes a complementary determining region
(CDR) that recognizes and binds to a specific binding site on a
particular antigen. The constant region, located on the tail of the
Y, is recognized by and interacts with the immune system (Janeway,
C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th
Ed., Garland Publishing, New York). A target antigen generally has
numerous binding sites, also called epitopes, recognized by CDRs on
multiple antibodies. Each antibody that specifically binds to a
different epitope has a different structure. Thus, one antigen may
have more than one corresponding antibody.
The term "antibody" as used herein, also refers to a full-length
immunoglobulin molecule or an immunologically active portion of a
full-length immunoglobulin molecule, i.e., a molecule that contains
an antigen binding site that immunospecifically binds an antigen of
a target of interest or part thereof, such targets including but
not limited to, cancer cell or cells that produce autoimmune
antibodies associated with an autoimmune disease. The
immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE,
IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule. The immunoglobulins
can be derived from any species. In one aspect, however, the
immunoglobulin is of human, murine, or rabbit origin. In another
aspect, the antibodies are polyclonal, monoclonal, bispecific,
human, humanized or chimeric antibodies, single chain antibodies,
Fv, Fab fragments, F(ab') fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, CDR's, and epitope-binding fragments of any
of the above which immunospecifically bind to cancer cell antigens,
viral antigens or microbial antigens.
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations which
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they may be synthesized uncontaminated by other antibodies. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.
(1975) Nature 256:495, or may be made by recombinant DNA methods
(see, U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may
also be isolated from phage antibody libraries using the techniques
described in Clackson et al. (1991) Nature, 352:624-628 and Marks
et al. (1991) J. Mol. Biol., 222:581-597, for example.
The monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl.
Acad. Sci. USA, 81:6851-6855).
Various methods have been employed to produce monoclonal antibodies
(MAbs). Hybridoma technology, which refers to a cloned cell line
that produces a single type of antibody, uses the cells of various
species, including mice (murine), hamsters, rats, and humans.
Another method to prepare MAbs uses genetic engineering including
recombinant DNA techniques. Monoclonal antibodies made from these
techniques include, among others, chimeric antibodies and humanized
antibodies. A chimeric antibody combines DNA encoding regions from
more than one type of species. For example, a chimeric antibody may
derive the variable region from a mouse and the constant region
from a human. A humanized antibody comes predominantly from a
human, even though it contains nonhuman portions. Like a chimeric
antibody, a humanized antibody may contain a completely human
constant region. But unlike a chimeric antibody, the variable
region may be partially derived from a human. The nonhuman,
synthetic portions of a humanized antibody often come from CDRs in
murine antibodies. In any event, these regions are crucial to allow
the antibody to recognize and bind to a specific antigen.
As noted, murine antibodies can be used. While useful for
diagnostics and short-term therapies, murine antibodies cannot be
administered to people long-term without increasing the risk of a
deleterious immunogenic response. This response, called Human
Anti-Mouse Antibody (HAMA), occurs when a human immune system
recognizes the murine antibody as foreign and attacks it. A HAMA
response can cause toxic shock or even death.
Chimeric and humanized antibodies reduce the likelihood of a HAMA
response by minimizing the nonhuman portions of administered
antibodies. Furthermore, chimeric and humanized antibodies have the
additional benefit of activating secondary human immune responses,
such as antibody dependent cellular cytotoxicity.
"Antibody fragments" comprise a portion of an intact antibody,
preferably comprising the antigen-binding or variable region
thereof. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragment(s).
An "intact" antibody is one which comprises an antigen-binding
variable region as well as a light chain constant domain (CL) and
heavy chain constant domains, CH1, CH2 and CH3. The constant
domains may be native sequence constant domains (e.g., human native
sequence constant domains) or amino acid sequence variant
thereof.
The intact antibody may have one or more "effector functions" which
refer to those biological activities attributable to the Fc region
(a native sequence Fc region or amino acid sequence variant Fc
region) of an antibody. Examples of antibody effector functions
include C1q binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g., B
cell receptor; BCR), etc.
Depending on the amino acid sequence of the constant domain of
their heavy chains, intact antibodies can be assigned to different
"classes." There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA,
and IgA2. The heavy-chain constant domains that correspond to the
different classes of antibodies are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
The expressions "ErbB2" and "HER2" are used interchangeably herein
and refer to human HER2 protein described, for example, in Semba et
al., Proc. Natl. Acad. Sci. USA, 82:6497-6501 (1985) and Yamamoto
et al., (1986) Nature, 319:230-234 (Genebank accession number
X03363). The term "erbB2" refers to the gene encoding human ErbB2
and "neu" refers to the gene encoding rat p185neu. Preferred ErbB2
is native sequence human ErbB2.
Antibodies to ErbB receptors are available commercially from a
number of sources, including, for example, Santa Cruz
Biotechnology, Inc., California, USA.
By "ErbB ligand" is meant a polypeptide which binds to and/or
activates an ErbB receptor. The ErbB ligand may be a native
sequence human ErbB ligand such as epidermal growth factor (EGF)
(Savage et al. (1972) J. Biol. Chem., 247:7612-7621); transforming
growth factor alpha (TGF-.alpha.) (Marquardt et al. (1984) Science
223:1079-1082); amphiregulin also known as schwanoma or
keratinocyte autocrine growth factor (Shoyab et al. (1989) Science
243:1074-1076; Kimura et al., Nature, 348:257-260 (1990); and Cook
et al., Mol. Cell. Biol., 11:2547-2557 (1991)); betacellulin (Shing
et al., Science, 259:1604-1607 (1993); and Sasada et al., Biochem.
Biophys. Res. Commun., 190:1173 (1993)); heparin-binding epidermal
growth factor (HB-EGF) (Higashiyama et al., Science, 251:936-939
(1991)); epiregulin (Toyoda et al., J. Biol. Chem., 270:7495-7500
(1995); and Komurasaki et al., Oncogene, 15:2841-2848 (1997)); a
heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al.,
Nature, 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al.,
Proc. Natl. Acad. Sci., 94:9562-9567 (1997)); neuregulin-4 (NRG-4)
(Harari et al., Oncogene, 18:2681-89 (1999)) or cripto (CR-1)
(Kannan et al., J. Biol. Chem., 272(6):3330-3335 (1997)). ErbB
ligands which bind EGFR include EGF, TGF-.alpha., amphiregulin,
betacellulin, HB-EGF and epiregulin. ErbB ligands which bind ErbB3
include heregulins. ErbB ligands capable of binding ErbB4 include
betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and
heregulins. The ErbB ligand may also be a synthetic ErbB ligand.
The synthetic ligand may be specific for a particular ErbB
receptor, or may recognize particular ErbB receptor complexes. An
example of a synthetic ligand is the synthetic heregulin/EGF
chimera biregulin (see, for example, Jones et al., (1999) FEBS
Letters, 447:227-231, which is incorporated by reference).
"Heregulin" (HRG) refers to a polypeptide encoded by the heregulin
gene product as disclosed in U.S. Pat. No. 5,641,869 or Marchionni
et al., Nature, 362:312-318 (1993). Examples of heregulins include
heregulin-.alpha., heregulin-.beta.1, heregulin-.beta.2 and
heregulin-.beta.3 (Holmes et al., Science, 256:1205-1210 (1992);
and U.S. Pat. No. 5,641,869); neu differentiation factor (NDF)
(Peles et al., Cell 69: 205-216 (1992)); acetylcholine
receptor-inducing activity (ARIA) (Falls et al. (1993) Cell
72:801-815); glial growth factors (GGFs) (Marchionni et al.,
Nature, 362:312-318 (1993)); sensory and motor neuron derived
factor (SMDF) (Ho et al., J. Biol. Chem., 270:14523-14532 (1995));
.gamma.-heregulin (Schaefer et al., Oncogene, 15:1385-1394 (1997)).
The term includes biologically active fragments and/or amino acid
sequence variants of a native sequence HRG polypeptide, such as an
EGF-like domain fragment thereof (e.g., HRG.beta.1177-244).
"ErbB hetero-oligomer" is a noncovalently associated oligomer
comprising at least two different ErbB receptors. An "ErbB dimer"
is a noncovalently associated oligomer that comprises two different
ErbB receptors. Such complexes may form when a cell expressing two
or more ErbB receptors is exposed to an ErbB ligand. ErbB
oligomers, such as ErbB dimers, can be isolated by
immunoprecipitation and analyzed by SDS-PAGE as described in
Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), for
example. Examples of such ErbB hetero-oligomers include EGFR-ErbB2
(also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and
ErbB3-ErbB4 (HER3/HER4) complexes. Moreover, the ErbB
hetero-oligomer may comprise two or more ErbB2 receptors combined
with a different ErbB receptor, such as ErbB3, ErbB4 or EGFR
(ErbB1). Other proteins, such as a cytokine receptor subunit (e.g.,
gp130) may be included in the hetero-oligomer.
A "native sequence" polypeptide is one which has the same amino
acid sequence as a polypeptide, e.g., tumor-associated antigen
receptor, derived from nature. Such native sequence polypeptides
can be isolated from nature or can be produced by recombinant or
synthetic means. Thus, a native sequence polypeptide can have the
amino acid sequence of naturally-occurring human polypeptide,
murine polypeptide, or polypeptide from any other mammalian
species.
The term "amino acid sequence variant" refers to polypeptides
having amino acid sequences that differ to some extent from a
native sequence polypeptide. Ordinarily, amino acid sequence
variants will possess at least about 70% homology with at least one
receptor binding domain of a native ligand, or with at least one
ligand binding domain of a native receptor, such as a
tumor-associated antigen, and preferably, they will be at least
about 80%, more preferably, at least about 90% homologous with such
receptor or ligand binding domains. The amino acid sequence
variants possess substitutions, deletions, and/or insertions at
certain positions within the amino acid sequence of the native
amino acid sequence.
"Sequence identity" is defined as the percentage of residues in the
amino acid sequence variant that are identical after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Methods and computer programs
for the alignment are well known in the art. One such computer
program is "Align 2," authored by Genentech, Inc., which was filed
with user documentation in the United States Copyright Office,
Washington, D.C. 20559, on Dec. 10, 1991.
"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to
a cell-mediated reaction in which nonspecific cytotoxic cells that
express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells,
neutrophils, and macrophages) recognize bound antibody on a target
cell and subsequently cause lysis of the target cell. The primary
cells for mediating ADCC, NK cells, express Fc.gamma.RIII only,
whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, (1991) Annu. Rev.
Immunol, 9:457-92. To assess ADCC activity of a molecule of
interest, an in vitro ADCC assay, such as that described in U.S.
Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector
cells for such assays include peripheral blood mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al., Proc. Natl. Acad. Sci. USA, 95:652-656 (1998).
The terms "Fc receptor" or "FcR" are used to describe a receptor
that binds to the Fc region of an antibody. The preferred FcR is a
native sequence human FcR. Moreover, a preferred FcR is one which
binds an IgG antibody (a gamma receptor) and includes receptors of
the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma. RIII subclasses,
including allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (See review M. in Daeron,
Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et
al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med., 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus. (Guyer
et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol.,
24:249 (1994)).
"Complement dependent cytotoxicity" or "CDC" refers to the ability
of a molecule to lyse a target in the presence of complement. The
complement activation pathway is initiated by the binding of the
first component of the complement system (C1q) to a molecule (e.g.,
an antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g., as described in Gazzano-Santoro et
al., J. Immunol. Methods, 202:163 (1996), may be performed.
The term "variable" refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies
and are used in the binding and specificity of each particular
antibody for its particular antigen. However, the variability is
not evenly distributed throughout the variable domains of
antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al. (1991) Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md.). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody dependent cellular cytotoxicity (ADCC).
The term "hypervariable region" when used herein refers to the
amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al. supra) and/or those
residues from a "hypervariable loop" (e.g., residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete
antigen-recognition and antigen-binding site. This region consists
of a dimer of one heavy chain and one light chain variable domain
in tight, non-covalent association. It is in this configuration
that the three hypervariable regions of each variable domain
interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six hypervariable regions confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three
hypervariable regions specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
The Fab fragment also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies from any vertebrate species can be
assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and
VL domains of antibody, wherein these domains are present in a
single polypeptide chain. Preferably, the Fv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two
antigen-binding sites, which fragments comprise a variable heavy
domain (VH) connected to a variable light domain (VL) in the same
polypeptide chain (VH-VL). By using a linker that is too short to
allow pairing between the two domains on the same chain, the
domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are
described more fully in, for example, EP 404,097; WO 93/11161; and
Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448.
"Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fe), typically that of a human
immunoglobulin. For further details, see Jones et al. (1986)
Nature, 321:522-525; Riechmann et al. (1988) Nature 332:323-329;
and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.
Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2,
huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and
huMAb4D5-8 (HERCEPTINI.RTM.) as described in Table 3 of U.S. Pat.
No. 5,821,337 expressly incorporated herein by reference; humanized
520C9 (WO 93/21319) and humanized 2C4 antibodies as described
herein below.
An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
An antibody "which binds" an antigen of interest is one capable of
binding that antigen with sufficient affinity such that the
antibody is useful in targeting a cell expressing the antigen.
An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is a tumor cell, e.g.,
a breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic or bladder cell. Various methods
are available for evaluating the cellular events associated with
apoptosis. For example, phosphatidyl serine (PS) translocation can
be measured by annexin binding; DNA fragmentation can be evaluated
through DNA laddering; and nuclear/chromatin condensation along
with DNA fragmentation can be evaluated by any increase in
hypodiploid cells.
A "disorder" is any condition that would benefit from treatment of
the present invention. This includes chronic and acute disorders or
diseases including those pathological conditions which predispose
the mammal to the disorder in question. Non-limiting examples of
disorders to be treated herein include benign and malignant tumors;
leukemia and lymphoid malignancies, in particular breast, ovarian,
stomach, endometrial, salivary gland, lung, kidney, colon, thyroid,
pancreatic, prostate or bladder cancer; neuronal, glial,
astrocytal, hypothalamic and other glandular, macrophagal,
epithelial, stromal and blastocoelic disorders; and inflammatory,
angiogenic and immunologic disorders.
The term "therapeutically effective amount" refers to an amount of
a drug effective to treat a disease or disorder in a mammal. In the
case of cancer, the therapeutically effective amount of the drug
may reduce the number of cancer cells; reduce the tumor size;
inhibit (i.e., slow to some extent and preferably stop) cancer cell
infiltration into peripheral organs; inhibit (i.e., slow to some
extent and preferably stop) tumor metastasis; inhibit, to some
extent, tumor growth; and/or relieve to some extent one or more of
the symptoms associated with the cancer. To the extent the drug may
prevent growth and/or kill existing cancer cells, it may be
cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for
example, be measured by assessing the time to disease progression
(TTP) and/or determining the response rate (RR).
The term "substantial amount" refers to a majority, i.e. >50% of
a population, of a collection or a sample.
The term "intracellular metabolite" refers to a compound resulting
from a metabolic process or reaction inside a cell on an antibody
drug conjugate (ADC). The metabolic process or reaction may be an
enzymatic process such as proteolytic cleavage of a peptide linker
of the ADC, or hydrolysis of a functional group such as a
hydrazone, ester, or amide. Intracellular metabolites include, but
are not limited to, antibodies and free drug which have undergone
intracellular cleavage after entry, diffusion, uptake or transport
into a cell.
The terms "intracellularly cleaved" and "intracellular cleavage"
refer to a metabolic process or reaction inside a cell on an
Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate, an antibody
drug conjugate (ADC) or the like whereby the covalent attachment,
e.g., the linker, between the drug moiety (D) and the antibody (Ab)
is broken, resulting in the free drug dissociated from the antibody
inside the cell. The cleaved moieties of the Drug-Ligand Conjugate,
a Drug-Linker-Ligand Conjugate or ADC are thus intracellular
metabolites.
The term "bioavailability" refers to the systemic availability
(i.e., blood/plasma levels) of a given amount of drug administered
to a patient. Bioavailability is an absolute term that indicates
measurement of both the time (rate) and total amount (extent) of
drug that reaches the general circulation from an administered
dosage form.
The term "cytotoxic activity" refers to a cell-killing, cytostatic
or anti-proliferation effect of an antibody drug conjugate compound
or an intracellular metabolite of an antibody drug conjugate
compound. Cytotoxic activity may be expressed as the IC.sub.50
value which is the concentration (molar or mass) per unit volume at
which half the cells survive.
The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells. Examples of cancer include, but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer including gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer.
An "ErbB2-expressing cancer" is one which produces sufficient
levels of ErbB2 at the surface of cells thereof, such that an
anti-ErbB2 antibody can bind thereto and have a therapeutic effect
with respect to the cancer.
A cancer "characterized by excessive activation" of an ErbB2
receptor is one in which the extent of ErbB2 receptor activation in
cancer cells significantly exceeds the level of activation of that
receptor in non-cancerous cells of the same tissue type. Such
excessive activation may result from overexpression of the ErbB2
receptor and/or greater than normal levels of an ErbB2 ligand
available for activating the ErbB2 receptor in the cancer cells.
Such excessive activation may cause and/or be caused by the
malignant state of a cancer cell. In some embodiments, the cancer
will be subjected to a diagnostic or prognostic assay to determine
whether amplification and/or overexpression of an ErbB2 receptor is
occurring which results in such excessive activation of the ErbB2
receptor. Alternatively, or additionally, the cancer may be
subjected to a diagnostic or prognostic assay to determine whether
amplification and/or overexpression an ErbB2 ligand is occurring in
the cancer which attributes to excessive activation of the
receptor. In a subset of such cancers, excessive activation of the
receptor may result from an autocrine stimulatory pathway.
A cancer which "overexpresses" an ErbB2 receptor is one which has
significantly higher levels of an ErbB2 receptor at the cell
surface thereof, compared to a noncancerous cell of the same tissue
type. Such overexpression may be caused by gene amplification or by
increased transcription or translation. ErbB2 receptor
overexpression may be determined in a diagnostic or prognostic
assay by evaluating increased levels of the ErbB2 protein present
on the surface of a cell (e.g., via an immunohistochemistry assay;
IHC). Alternatively, or additionally, one may measure levels of
ErbB2-encoding nucleic acid in the cell, e.g., via fluorescent in
situ hybridization (FISH; see WO 98/45479), southern blotting, or
polymerase chain reaction (PCR) techniques, such as real time
quantitative PCR (RT-PCR). Overexpression of the ErbB2 ligand, may
be determined diagnostically by evaluating levels of the ligand (or
nucleic acid encoding it) in the patient, e.g., in a tumor biopsy
or by various diagnostic assays such as the IHC, FISH, southern
blotting, PCR or in vivo assays described above. One may also study
ErbB2 receptor overexpression by measuring shed antigen (e.g.,
ErbB2 extracellular domain) in a biological fluid such as serum
(see, e.g., U.S. Pat. No. 4,933,294; WO 91/05264; U.S. Pat. No.
5,401,638; and Sias et al., (1990) J. Immunol. Methods, 132:
73-80). Aside from the above assays, various other in vivo assays
are available to the skilled practitioner. For example, one may
expose cells within the body of the patient to an antibody which is
optionally labeled with a detectable label, e.g., a radioactive
isotope, and binding of the antibody to cells in the patient can be
evaluated, e.g., by external scanning for radioactivity or by
analyzing a biopsy taken from a patient previously exposed to the
antibody.
The tumors overexpressing HER2 are rated by immunohistochemical
scores corresponding to the number of copies of HER2 molecules
expressed per cell, and can been determined biochemically:
0=0-10,000 copies/cell, 1+=at least about 200,000 copies/cell,
2+=at least about 500,000 copies/cell, 3+=about 1-2.times.10.sup.6
copies/cell. Overexpression of HER2 at the 3+ level, which leads to
ligand-independent activation of the tyrosine kinase (Hudziak et
al., (1987) Proc. Natl. Acad. Sci. USA, 84:7159-7163), occurs in
approximately 30% of breast cancers, and in these patients,
relapse-free survival and overall survival are diminished (Slamon
et al., (1989) Science, 244:707-712; Slamon et al., (1987) Science,
235:177-182).
Conversely, a cancer which is "not characterized by overexpression
of the ErbB2 receptor" is one which, in a diagnostic assay, does
not express higher than normal levels of ErbB2 receptor compared to
a noncancerous cell of the same tissue type.
The term "cytotoxic agent" as used herein refers to a substance
that inhibits or prevents the function of cells and/or causes
destruction of cells. The term is intended to include radioactive
isotopes (e.g., .sup.211A, .sup.131I, .sup.125I, .sup.90Y,
.sup.186Re, .sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P, .sup.60C,
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including synthetic
analogs and derivatives thereof. In one aspect, the term is not
intended to include radioactive isotopes.
A "chemotherapeutic agent" is a chemical compound useful in the
treatment of cancer. Examples of chemotherapeutic agents include
alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; TLK 286 (TELCYTA.TM.); acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; bisphosphonates, such as
clodronate; antibiotics such as the enediyne antibiotics (e.g.,
calicheamicin, especially calicheamicin gamma1I and calicheamicin
omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186
(1994)) and anthracyclines such as annamycin, AD 32, alcarubicin,
daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100,
idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A,
an esperamicin, neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores, aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal
doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, and zorubicin; folic acid analogues such as denopterin,
pteropterin, and trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs
such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, and
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, and testolactone;
anti-adrenals such as aminoglutethimide, mitotane, and trilostane;
folic acid replenisher such as folinic acid (leucovorin);
aceglatone; anti-folate anti-neoplastic agents such as ALIMTA.RTM.,
LY231514 pemetrexed, dihydrofolate reductase inhibitors such as
methotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and
its prodrugs such as UFT, S-1 and capecitabine, and thymidylate
synthase inhibitors and glycinamide ribonucleotide
formyltransferase inhibitors such as raltitrexed (TOMUDEX.RTM.,
TDX); inhibitors of dihydropyrimidine dehydrogenase such as
eniluracil; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate; an
epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins;
mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;
phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g.,
TAXOL.RTM. paclitaxel (Bristol-Myers Squibb Oncology, Princeton,
N.J.), ABRAXANE.TM. Cremophor-free, albumin-engineered nanoparticle
formulation of paclitaxel (American Pharmaceutical Partners,
Schaumberg, Ill.), and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc
Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR.RTM.);
6-thioguanine; mercaptopurine; platinum; platinum analogs or
platinum-based analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine (VELBAN.RTM.); etoposide (VP-16);
ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.); vinca
alkaloid; vinorelbine (NAVELBINE.RTM.); novantrone; edatrexate;
daunomycin; aminopterin; xeloda; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone, and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and
leucovorin.
Also included in this definition are anti-hormonal agents that act
to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON.RTM.
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras,
and epidermal growth factor receptor (EGF-R); vaccines such as gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; PROLEUKIN.RTM.
rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; and pharmaceutically acceptable salts, acids or derivatives
of any of the above.
As used herein, the term "EGFR-targeted drug" refers to a
therapeutic agent that binds to EGFR and, optionally, inhibits EGFR
activation. Examples of such agents include antibodies and small
molecules that bind to EGFR. Examples of antibodies which bind to
EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),
MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat.
No. 4,943,533, Mendelsohn et al.) and variants thereof, such as
chimerized 225 (C225 or Cetuximab; ERBITUX.RTM.) and reshaped human
225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies
that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized
and chimeric antibodies that bind EGFR as described in U.S. Pat.
No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF
(see WO 98/50433, Abgenix). The anti-EGFR antibody may be
conjugated with a cyotoxic agent, thus generating an
immunoconjugate (see, e.g., EP 659,439A2, Merck Patent GmbH).
Examples of small molecules that bind to EGFR include ZD1839 or
Gefitinib (IRESSATMi; Astra Zeneca), Erlotinib HCl (CP-358774,
TARCEVA.TM.; Genentech/OSI) and AG1478, AG1571 (SU 5271;
Sugen).
A "tyrosine kinase inhibitor" is a molecule which inhibits to some
extent tyrosine kinase activity of a tyrosine kinase such as an
ErbB receptor. Examples of such inhibitors include the
EGFR-targeted drugs noted in the preceding paragraph as well as
quinazolines such as PD 153035, 4-(3-chloroanilino) quinazoline,
pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as
CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lambert);
antisense molecules (e.g., those that bind to ErbB-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate
(Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);
CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474
(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone);
or as described in any of the following patent publications: U.S.
Pat. No. 5,804,396; WO 99/09016 (American Cyanamid); WO 98/43960
(American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378
(Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347
(Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO
96/33980 (Zeneca).
An "anti-angiogenic agent" refers to a compound which blocks, or
interferes with to some degree, the development of blood vessels.
The anti-angiogenic factor may, for instance, be a small molecule
or antibody that binds to a growth factor or growth factor receptor
involved in promoting angiogenesis. In one embodiment, the
anti-angiogenic factor is an antibody that binds to Vascular
Endothelial Growth Factor (VEGF).
The term "cytokine" is a generic term for proteins released by one
cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically or hydrolytically activated
or converted into the more active parent form. See, e.g., Wilman,
"Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions,
14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al.,
"Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed
Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as including the anti-CD30, CD40, CD70 or
Lewis Y antibodies and, optionally, a chemotherapeutic agent) to a
mammal. The components of the liposome are commonly arranged in a
bilayer formation, similar to the lipid arrangement of biological
membranes.
The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
An "isolated" nucleic acid molecule is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural
source of the antibody nucleic acid. An isolated nucleic acid
molecule is other than in the form or setting in which it is found
in nature. Isolated nucleic acid molecules therefore are
distinguished from the nucleic acid molecule as it exists in
natural cells. However, an isolated nucleic acid molecule includes
a nucleic acid molecule contained in cells that ordinarily express
the antibody where, for example, the nucleic acid molecule is in a
chromosomal location different from that of natural cells.
The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking can be accomplished
by ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers can be
used in accordance with conventional practice.
As used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells"
include the primary subject cell and cultures derived therefrom
without regard for the number of transfers. It is also understood
that all progeny may not be precisely identical in DNA content, due
to deliberate or inadvertent mutations. Mutant progeny that have
the same function or biological activity as screened for in the
originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
An "autoimmune disease" herein is a disease or disorder arising
from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and
ankylosing spondylitis), psoriasis, dermatitis including atopic
dermatitis; chronic idiopathic urticaria, including chronic
autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal
necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease (IBD) (Crohn's disease,
ulcerative colitis), and IBD with co-segregate of pyoderma
gangrenosum, erythema nodosum, primary sclerosing cholangitis,
and/or episcleritis), respiratory distress syndrome, including
adult respiratory distress syndrome (ARDS), meningitis,
IgE-mediated diseases such as anaphylaxis and allergic rhinitis,
encephalitis such as Rasmussen's encephalitis, uveitis, colitis
such as microscopic colitis and collagenous colitis,
glomerulonephritis (GN) such as membranous GN, idiopathic
membranous GN, membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
eczema, asthma, conditions involving infiltration of T cells and
chronic inflammatory responses, atherosclerosis, autoimmune
myocarditis, leukocyte adhesion deficiency, systemic lupus
erythematosus (SLE) such as cutaneous SLE, lupus (including
nephritis, cerebritis, pediatric, non-renal, discoid, alopecia),
juvenile onset diabetes, multiple sclerosis (MS) such as
spino-optical MS, allergic encephalomyelitis, immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including Wegener's granulomatosis, agranulocytosis,
vasculitis (including Large Vessel vasculitis (including
Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis),
Medium Vessel vasculitis (including Kawasaki's Disease and
Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated
vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS),
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
immune hemolytic anemia including autoimmune hemolytic anemia
(AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome,
myasthenia gravis, antigen-antibody complex mediated diseases,
anti-glomerular basement membrane disease, anti-phospholipid
antibody syndrome, allergic neuritis, Bechet disease, Castleman's
syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic
Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson
syndrome, solid organ transplant rejection (including pretreatment
for high panel reactive antibody titers, IgA deposit in tissues,
and rejection arising from renal transplantation, liver
transplantation, intestinal transplantation, cardiac
transplantation, etc.), graft versus host disease (GVHD),
pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and
pemphigus mucus-membrane pemphigoid), autoimmune
polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune
complex nephritis, IgM polyneuropathies or IgM mediated neuropathy,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), thrombocytopenia (as developed by
myocardial infarction patients, for example), including autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimmune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM),
including pediatric IDDM, and Sheehan's syndrome; autoimmune
hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis
obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome,
Berger's Disease (IgA nephropathy), primary biliary cirrhosis,
celiac sprue (gluten enteropathy), refractory sprue with
co-segregate dermatitis herpetiformis, cryoglobulinemia,
amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease),
coronary artery disease, autoimmune inner ear disease (AIED),
autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS),
polychondritis such as refractory polychondritis, pulmonary
alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,
monoclonal gammopathy of uncertain/unknown significance (MGUS),
peripheral neuropathy, paraneoplastic syndrome, channelopathies
such as epilepsy, migraine, arrhythmia, muscular disorders,
deafness, blindness, periodic paralysis, and channelopathies of the
CNS; autism, inflammatory myopathy, and focal segmental
glomerulosclerosis (FSGS).
"Alkyl" is C.sub.1-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms. Examples are methyl
(Me, --CH.sub.3), ethyl (Et, --CH.sub.2CH.sub.3), 1-propyl (n-Pr,
n-propyl, --CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (1-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3.
"Alkenyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp.sup.2 double bond.
Examples include, but are not limited to: ethylene or vinyl
(--CH.dbd.CH.sub.2), allyl (--CH.sub.2CH.dbd.CH.sub.2),
cyclopentenyl (--C.sub.5H.sub.7), and 5-hexenyl (--CH.sub.2
CH.sub.2CH.sub.2CH.sub.2CH.dbd.CH.sub.2).
"Alkynyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp triple bond. Examples
include, but are not limited to: acetylenic (--C.ident.CH) and
propargyl (--CH.sub.2C.ident.CH).
"Alkylene" refers to a saturated, branched or straight chain or
cyclic hydrocarbon radical of 1-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkane. Typical alkylene radicals include, but are not limited to:
methylene (--CH.sub.2--) 1,2-ethyl (--CH.sub.2CH.sub.2--),
1,3-propyl (--CH.sub.2CH.sub.2CH.sub.2--), 1,4-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like.
"Alkenylene" refers to an unsaturated, branched or straight chain
or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkene. Typical alkenylene radicals include, but are not limited
to: 1,2-ethylene (--CH.dbd.CH--).
"Alkynylene" refers to an unsaturated, branched or straight chain
or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkyne. Typical alkynylene radicals include, but are not limited
to: acetylene propargyl (--CH.sub.2C.dbd.C--), and 4-pentynyl
(--CH.sub.2CH.sub.2CH.sub.2C.ident.CH--).
"Aryl" means a monovalent aromatic hydrocarbon radical of 6-20
carbon atoms derived by the removal of one hydrogen atom from a
single carbon atom of a parent aromatic ring system. Some aryl
groups are represented in the exemplary structures as "Ar". Typical
aryl groups include, but are not limited to, radicals derived from
benzene, substituted benzene, naphthalene, anthracene, biphenyl,
and the like.
"Arylalkyl" refers to an acyclic alkyl radical in which one of the
hydrogen atoms bonded to a carbon atom, typically a terminal or
sp.sup.3 carbon atom, is replaced with an aryl radical. Typical
arylalkyl groups include, but are not limited to, benzyl,
2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,
2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,
2-naphthophenylethan-1-yl and the like. The arylalkyl group
comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including
alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to
6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
"Heteroarylalkyl" refers to an acyclic alkyl radical in which one
of the hydrogen atoms bonded to a carbon atom, typically a terminal
or sp.sup.3 carbon atom, is replaced with a heteroaryl radical.
Typical heteroarylalkyl groups include, but are not limited to,
2-benzimidazolylmethyl, 2-furylethyl, and the like. The
heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g., the
alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the
heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl
moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from
N, O, P, and S. The heteroaryl moiety of the heteroarylalkyl group
may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and S), for example: a
bicyclo [4,5], [5,5], [5,6], or [6,6] system.
"Substituted alkyl", "substituted aryl", and "substituted
arylalkyl" mean alkyl, aryl, and arylalkyl respectively, in which
one or more hydrogen atoms are each independently replaced with a
substituent. Typical substituents include, but are not limited to,
--X, --R, --O.sup.-, --OR, --SR, --S.sup.-, --NR.sub.2, --NR.sub.3,
.dbd.NR, --CX.sub.3, --CN, --OCN, --SCN, --N.dbd.C.dbd.O, --NCS,
--NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3, NC(.dbd.O)R,
--C(.dbd.O)R, --C(.dbd.O)NR.sub.2, --SO.sub.3.sup.-, --SO.sub.3H,
--S(.dbd.O).sub.2R, --OS(.dbd.O).sub.2OR, --S(.dbd.O).sub.2NR,
--S(.dbd.O)R, --OP(.dbd.O)(OR).sub.2, --P(.dbd.O)(OR).sub.2,
--PO.sup.-.sub.3, --PO.sub.3H.sub.2, --C(.dbd.O)R, --C(.dbd.O)X,
--C(.dbd.S)R, --CO.sub.2R, --CO.sub.2.sup.-, --C(.dbd.S)OR,
--C(.dbd.O)SR, --C(.dbd.S)SR, --C(.dbd.O)NR.sub.2,
--C(.dbd.S)NR.sub.2, --C(.dbd.NR)NR.sub.2, where each X is
independently a halogen: F, Cl, Br, or I; and each R is
independently --H, C.sub.2-C.sub.18 alkyl, C.sub.6-C.sub.20 aryl,
C.sub.3-C.sub.14 heterocycle, protecting group or prodrug moiety.
Alkylene, alkenylene, and alkynylene groups as described above may
also be similarly substituted.
"Heteroaryl" and "Heterocycle" refer to a ring system in which one
or more ring atoms is a heteroatom, e.g., nitrogen, oxygen, and
sulfur. The heterocycle radical comprises 1 to 20 carbon atoms and
1 to 3 heteroatoms selected from N, O, P, and S. A heterocycle may
be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and
1 to 3 heteroatoms selected from N, O, P, and S) or a bicycle
having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3
heteroatoms selected from N, O, P, and S), for example: a
bicyclo[4,5], [5,5], [5,6], or [6,6] system.
Heterocycles are described in Paquette, Leo A.; "Principles of
Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968),
particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of
Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,
19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
Examples of heterocycles include by way of example and not
limitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl),
thiazolyl, tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,
pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,
indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl,
pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,
tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl.
By way of example and not limitation, carbon bonded heterocycles
are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3,
4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine,
position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a
furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4
of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more
typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,
4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,
5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,
5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or
5-thiazolyl.
By way of example and not limitation, nitrogen bonded heterocycles
are bonded at position 1 of an aziridine, azetidine, pyrrole,
pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,
2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,
3-pyrazoline, piperidine, piperazine, indole, indoline,
1H-indazole, position 2 of a isoindole, or isoindoline, position 4
of a morpholine, and position 9 of a carbazole, or
.beta.-carboline. Still more typically, nitrogen bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
"Carbocycle" means a saturated or unsaturated ring having 3 to 7
carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.
Monocyclic carbocycles have 3 to 6 ring atoms, still more typically
5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms,
e.g., arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or
9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system.
Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl,
cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and
cyclooctyl.
"Linker", "Linker Unit", or "link" means a chemical moiety
comprising a covalent bond or a chain of atoms that covalently
attaches an antibody to a drug moiety. In various embodiments, a
linker is specified as LU. Linkers include a divalent radical such
as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:
--(CR.sub.2).sub.nO(CR.sub.2).sub.n--, repeating units of alkyloxy
(e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.,
polyethyleneamino, Jeffamine.TM.); and diacid ester and amides
including succinate, succinamide, diglycolate, malonate, and
caproamide.
The term "chiral" refers to molecules which have the property of
non-superimposability of the mirror image partner, while the term
"achiral" refers to molecules which are superimposable on their
mirror image partner.
The term "stereoisomers" refers to compounds which have identical
chemical constitution, but differ with regard to the arrangement of
the atoms or groups in space.
"Diastereomer" refers to a stereoisomer with two or more centers of
chirality and whose molecules are not mirror images of one another.
Diastereomers have different physical properties, e.g., melting
points, boiling points, spectral properties, and reactivities.
Mixtures of diastereomers may separate under high resolution
analytical procedures such as electrophoresis and
chromatography.
"Enantiomers" refer to two stereoisomers of a compound which are
non-superimposable mirror images of one another.
Stereochemical definitions and conventions used herein generally
follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms
(1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen,
S., Stereochemistry of Organic Compounds (1994) John Wiley &
Sons, Inc., New York. Many organic compounds exist in optically
active forms, i.e., they have the ability to rotate the plane of
plane-polarized light. In describing an optically active compound,
the prefixes D and L, or R and S, are used to denote the absolute
configuration of the molecule about its chiral center(s). The
prefixes d and 1 or (+) and (-) are employed to designate the sign
of rotation of plane-polarized light by the compound, with (-) or 1
meaning that the compound is levorotatory. A compound prefixed with
(+) or d is dextrorotatory. For a given chemical structure, these
stereoisomers are identical except that they are mirror images of
one another. A specific stereoisomer may also be referred to as an
enantiomer, and a mixture of such isomers is often called an
enantiomeric mixture. A 50:50 mixture of enantiomers is referred to
as a racemic mixture or a racemate, which may occur where there has
been no stereoselection or stereospecificity in a chemical reaction
or process. The terms "racemic mixture" and "racemate" refer to an
equimolar mixture of two enantiomeric species, devoid of optical
activity.
Examples of a "patient" include, but are not limited to, a human,
rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat,
bird and fowl. In an exemplary embodiment, the patient is a
human.
"Aryl" refers to a carbocyclic aromatic group. Examples of aryl
groups include, but are not limited to, phenyl, naphthyl and
anthracenyl. A carbocyclic aromatic group or a heterocyclic
aromatic group can be unsubstituted or substituted with one or more
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
The term "C.sub.1-C.sub.8 alkyl," as used herein refers to a
straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 8 carbon atoms. Representative "C.sub.1-C.sub.8
alkyl" groups include, but are not limited to, -methyl, -ethyl,
-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl,
-n-nonyl and -n-decyl; while branched C.sub.1-C.sub.8 alkyls
include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,
-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C.sub.1-C.sub.8
alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl,
-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,
1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl,
-2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl. methyl,
ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, text-butyl,
n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl,
3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl,
2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl,
2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl,
isoheptyl, n-octyl, and isooctyl. A C.sub.1-C.sub.8 alkyl group can
be unsubstituted or substituted with one or more groups including,
but not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --SO.sub.3R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; where each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
A "C.sub.3-C.sub.8 carbocycle" is a 3-, 4-, 5-, 6-, 7- or
8-membered saturated or unsaturated non-aromatic carbocyclic ring.
Representative C.sub.3-C.sub.8 carbocycles include, but are not
limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl,
-cyclopentadienyl, -cyclohexyl, -cyclohexenyl,
-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,
-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and
-cyclooctadienyl. A C.sub.3-C.sub.3 carbocycle group can be
unsubstituted or substituted with one or more groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; where each R' is independently selected from H,
--C.sub.1-C.sub.8 alkyl and aryl.
A "C.sub.3-C.sub.8 carbocyclo" refers to a C.sub.3-C.sub.8
carbocycle group defined above wherein one of the carbocycle
groups' hydrogen atoms is replaced with a bond.
A "C.sub.1-C.sub.10 alkylene" is a straight chain, saturated
hydrocarbon group of the formula --(CH.sub.2).sub.1-10--. Examples
of a C.sub.1-C.sub.10 alkylene include methylene, ethylene,
propylene, butylene, pentylene, hexylene, heptylene, ocytylene,
nonylene and decalene.
An "arylene" is an aryl group which has two covalent bonds and can
be in the ortho, meta, or para configurations as shown in the
following structures:
##STR00005## in which the phenyl group can be unsubstituted or
substituted with up to four groups including, but not limited to,
--C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R', --OH,
-halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and --CN;
wherein each R' is independently selected from H, --C.sub.1-C.sub.8
alkyl and aryl.
A "C.sub.3-C.sub.8 heterocycle" refers to an aromatic or
non-aromatic C.sub.3-C.sub.8 carbocycle in which one to four of the
ring carbon atoms are independently replaced with a heteroatom from
the group consisting of O, S and N. Representative examples of a
C.sub.3-C.sub.8 heterocycle include, but are not limited to,
benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl,
isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,
imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl,
pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl,
isoxazolyl and tetrazolyl. A C.sub.3-C.sub.8 heterocycle can be
unsubstituted or substituted with up to seven groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; wherein each R' is independently selected from H,
--C.sub.1-C.sub.8 alkyl and aryl.
"C.sub.3-C.sub.8 heterocyclo" refers to a C.sub.3-C.sub.8
heterocycle group defined above wherein one of the heterocycle
group's hydrogen atoms is replaced with a bond. A C.sub.3-C.sub.8
heterocyclo can be unsubstituted or substituted with up to six
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
An "Exemplary Compound" is a Drug Compound or a Drug-Linker
Compound.
An "Exemplary Conjugate" is a Drug-Ligand Conjugate having a
cleavable Drug unit from the Drug-Ligand Conjugate or a
Drug-Linker-Ligand Conjugate.
In some embodiments, the Exemplary Compounds and Exemplary
Conjugates are in isolated or purified form. As used herein,
"isolated" means separated from other components of (a) a natural
source, such as a plant or animal cell or cell culture, or (b) a
synthetic organic chemical reaction mixture. As used herein,
"purified" means that when isolated, the isolate contains at least
95%, and in another aspect at least 98%, of Exemplary Compound or
Exemplary Conjugate by weight of the isolate.
Examples of a "hydroxylprotecting group" include, but are not
limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether,
tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether,
trimethylsilyl ether, triethylsilyl ether, triisopropyl silyl
ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether,
acetate ester, substituted acetate esters, pivaloate, benzoate,
methanesulfonate and p-toluenesulfonate.
"Leaving group" refers to a functional group that can be
substituted by another functional group. Such leaving groups are
well known in the art, and examples include, but are not limited
to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl
(mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl
(triflate), and trifluoromethylsulfonate.
The phrase "pharmaceutically acceptable salt," as used herein,
refers to pharmaceutically acceptable organic or inorganic salts of
an Exemplary Compound or Exemplary Conjugate. The Exemplary
Compounds and Exemplary Conjugates contain at least one amino
group, and accordingly acid addition salts can be formed with this
amino group. Exemplary salts include, but are not limited, to
sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,
nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,
lactate, salicylate, acid citrate, tartrate, oleate, tannate,
pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counterion. The counterion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counterion.
"Pharmaceutically acceptable solvate" or "solvate" refer to an
association of one or more solvent molecules and a compound of the
invention, e.g., an Exemplary Compound or Exemplary Conjugate.
Examples of solvents that form pharmaceutically acceptable solvates
include, but are not limited to, water, isopropanol, ethanol,
methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
The following abbreviations are used herein and have the indicated
definitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit
is citrulline, dap is dolaproine, DCC is
1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is
diethylamine, DEAD is diethylazodicarboxylate, DEPC is
diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate,
DIEA is N,N-diisopropylethylamine, dil is dolaisoleuine, DMAP is
4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or
1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is
dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline,
DTNB is 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is
diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ
is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is
electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is
N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high
pressure liquid chromatography, ile is isoleucine, lys is lysine,
MeCN(CH.sub.3CN) is acetonitrile, MeOH is methanol, Mtr is
4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is
(1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is
phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph
is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe is
L-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphonium
hexafluorophosphate, SEC is size-exclusion chromatography, Su is
succinimide, TBTU is O-benzotriazol-1-yl-N,N,N,N-tetramethyluronium
tetrafluoroborate, TFA is trifluoroacetic acid, TLC is thin layer
chromatography, UV is ultraviolet, and val is valine.
The following linker abbreviations are used herein and have the
indicated definitions: Val Cit is a valine-citrulline, dipeptide
site in protease cleavable linker; PAB is p-aminobenzylcarbamoyl;
(Me)vc is N-methyl-valine citrulline, where the linker peptide bond
has been modified to prevent its cleavage by cathepsin B;
MC(PEG)6-OH is maleimidocaproyl-polyethylene glycol; SPP is
N-Succinimidyl 4-(2-pyridylthio) pentanoate; and SMCC is
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate.
The terms "treat" or "treatment," unless otherwise indicated by
context, refer to both therapeutic treatment and prophylactic or
preventative measures, wherein the object is to prevent or slow
down (lessen) an undesired physiological change or disorder, such
as the development or spread of cancer. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. Those in need of treatment include those
already with the condition or disorder as well as those prone to
have the condition or disorder or those in which the condition or
disorder is to be prevented.
In the context of cancer, the term "treating" includes any or all
of preventing growth of tumor cells, cancer cells, or of a tumor;
preventing replication of tumor cells or cancer cells, lessening of
overall tumor burden or decreasing the number of cancerous cells,
and ameliorating one or more symptoms associated with the
disease.
In the context of an autoimmune disease, the term "treating"
includes any or all of preventing replication of cells associated
with an autoimmune disease state including, but not limited to,
cells that produce an autoimmune antibody, lessening the
autoimmune-antibody burden and ameliorating one or more symptoms of
an autoimmune disease.
In the context of an infectious disease, the term "treating"
includes any or all of: preventing the growth, multiplication or
replication of the pathogen that causes the infectious disease and
ameliorating one or more symptoms of an infectious disease.
The following cytotoxic drug abbreviations are used herein and have
the indicated definitions: MMAE is mono-methyl auristatin E (MW
718); MMAF is
N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine (MW
731.5); MMAF-DMAEA is MMAF with DMAEA (dimethylaminoethylamine) in
an amide linkage to the C-terminal phenylalanine (MW 801.5);
MMAF-TEG is MMAF with tetraethylene glycol esterified to the
phenylalanine; MMAF-NtBu is N-t-butyl, attached as an amide to
C-terminus of MMAF; AEVB is auristatin E valeryl benzylhydrazone,
acid labile linker through the C-terminus of AE (MW 732); and AFP
is Monoamide of p-phenylene diamine with C-terminal Phenylalanine
of Auristatin F (MW 732).
4.2 The Compounds of the Invention
4.2.1 The Compounds of Formula (Ia)
In one aspect, the invention provides Drug-Linker-Ligand Conjugates
having Formula Ia: LA.sub.a-W.sub.w--Y.sub.y-D).sub.p Ia
or a pharmaceutically acceptable salt or solvate thereof
wherein,
L- is a Ligand unit;
-A.sub.a-W.sub.w--Y.sub.y-- is a Linker unit (LU), wherein the
Linker unit includes:
-A- is a Stretcher unit,
a is 0 or 1,
each --W-- is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
--Y-- is a Spacer unit, and
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the Formulas D.sub.E and D.sub.F:
##STR00006##
wherein, independently at each location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH;
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
n is an integer ranging from 0 to 6.
In another embodiment, the present invention provides Drug
Compounds having the Formula Ib:
##STR00007## or pharmaceutically acceptable salts or solvates
thereof,
wherein:
R.sup.2 is selected from hydrogen and --C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 jointly, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are
attached;
R.sup.6 is selected from H and --C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, --OH,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl);
R.sup.9 is selected from H and --C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl group or --C.sub.3-C.sub.8
heterocycle;
Z is --O--, S, NH, or --NR.sup.12--, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
--C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is --C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or --C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, --C.sub.1-C.sub.8
alkyl, or --(CH.sub.2), --COOH; and
n is an integer ranging from 0 to 6.
In yet another embodiment, the invention provides
Drug-Linker-Ligand Conjugates having the Formula Ia':
AbA.sub.a-W.sub.w--Y.sub.y-D).sub.p Formula Ia' or pharmaceutically
acceptable salts or solvates thereof.
wherein:
Ab is an antibody,
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a Drug moiety selected from Formulas D.sub.E and D.sub.F:
##STR00008##
wherein, independently at each location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH;
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
n is an integer ranging from 0 to 6.
Ab is any antibody covalently attached to one or more drug units.
Ab includes an antibody which binds to CD30, CD40, CD70, Lewis Y
antigen. In another embodiment, Ab does not include an antibody
which binds to an ErbB receptor or to one or more of receptors
(1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank
accession no. NM.sub.--001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.--003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate,
Genbank accession no. NM.sub.--012449);
(4) 0772P(CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
(11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no.
AF455138);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor
potential cation channel, subfamily M, member 4, Genbank accession
no. NM.sub.--017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived
growth factor, Genbank accession no. NP.sub.--003203 or
NM.sub.--003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr
virus receptor) or Hs.73792, Genbank accession no. M26004);
(15) CD79b (1 Gb (immunoglobulin-associated beta), B29, Genbank
accession no. NM.sub.--000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase
anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no.
NM.sub.--030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20R.alpha. (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM.sub.--004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763);
(26) BAFF-R (Genbank accession no. NP.sub.--443177.1);
(27) CD22 (Genbank accession no. NP-001762.1);
(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation, Genbank
accession No. NP.sub.--001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled
receptor that is activated by the CXCL13 chemokine, functions in
lymphocyte migration and humoral defense, plays a role in HIV-2
infection and perhaps development of AIDS, lymphoma, myeloma, and
leukemia, Genbank accession No. NP.sub.--001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)
that binds peptides and presents them to CD4+ T lymphocytes,
Genbank accession No. NP.sub.--002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an
ion channel gated by extracellular ATP, may be involved in synaptic
transmission and neurogenesis, deficiency may contribute to the
pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank
accession No. NP.sub.--001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the
immunoglobulin Fc domain that contains C2 type Ig-like and ITAM
domains, may have a role in B-lymphocyte differentiation, Genbank
accession No. NP.sub.--443170.1); and/or
(35) IRTA2 (Immunoglobulin superfamily receptor translocation
associated 2, a putative immunoreceptor with possible roles in B
cell development and lymphomagenesis; deregulation of the gene by
translocation occurs in some B cell malignancies, Genbank accession
No. NP.sub.--112571.1).
In one embodiment --Ww- is -Val-Cit-.
In another embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl. In yet another
embodiment, R.sup.2 and R.sup.6 are each methyl, and R.sup.9 is
--H.
In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
In an exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H, R.sup.7 is
sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3, and R.sup.9
is --H.
In one embodiment, Z is --O-- or --NH--.
In one embodiment, R.sup.10 is aryl
In an exemplary embodiment, R.sup.10 is -phenyl.
In an exemplary embodiment, when Z is --O--, R.sup.11 is --H,
methyl or t-butyl.
In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
In one aspect, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, hAC10, hBR96,
hS2C6, h1F6, and h2F2.
Exemplary embodiments of Formula Ia have the following
structures:
##STR00009## wherein L is an antibody, Val is valine, and Cit is
citrulline.
The drug loading is represented by p, the average number of drug
molecules per antibody in a molecule (e.g., of Formula Ia, Ia' and
Ic). Drug loading may range from 1 to 20 drugs (D) per Ligand
(e.g., Ab or mAb). Compositions of Formula Ia and Formula Ia'
include collections of antibodies conjugated with a range of drugs,
from 1 to 20. The average number of drugs per antibody in
preparation of conjugation reactions may be characterized by
conventional means such as mass spectroscopy, ELISA assay, and
HPLC. The quantitative distribution of Ligand-Drug-Conjugates in
terms of p may also be determined. In some instances, separation,
purification, and characterization of homogeneous
Ligand-Drug-conjugates where p is a certain value from
Ligand-Drug-Conjugates with other drug loadings may be achieved by
means such as reverse phase HPLC or electrophoresis.
4.2.2 The Drug Compounds of Formula (Ib)
In another aspect, the present invention provides Drug Compounds
having the Formula (Ib):
##STR00010##
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R.sup.2 is selected from -hydrogen and --C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from -hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from -hydrogen, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, -aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from --H and -methyl; or R.sup.4 and R.sup.5 jointly, have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the carbon atom to which they are
attached;
R.sup.6 is selected from --H and --C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.3-C.sub.8 carbocycle, aryl, --C.sub.1-C.sub.8 alkyl-aryl,
--C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 carbocycle),
--C.sub.3-C.sub.8 heterocycle and --C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from --H, --OH,
--C.sub.1-C.sub.8 alkyl, --C.sub.3-C.sub.8 carbocycle and
--O--(C.sub.1-C.sub.8 alkyl);
R.sup.9 is selected from --H and --C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl group or --C.sub.3-C.sub.8
heterocycle;
Z is --O--, --S--, --NH--, or --NR.sup.12--, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl;
R.sup.11 is selected from --H, C.sub.1-C.sub.20 alkyl, aryl,
--C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is --C.sub.2-C.sub.8 alkyl;
R.sup.14 is --H or --C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently --H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently --H, --C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH; and
n is an integer ranging from 0 to 6.
In one embodiment, R.sup.3, R.sup.4 and R.sup.7 are independently
isopropyl or sec-butyl and R.sup.5 is --H. In an exemplary
embodiment, R.sup.3 and R.sup.4 are each isopropyl, R.sup.5 is --H,
and R.sup.7 is sec-butyl.
In another embodiment, R.sup.2 and R.sup.6 are each methyl, and
R.sup.9 is --H.
In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
In an exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H, R.sup.7 is
sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3, and R.sup.9
is --H.
In one embodiment, Z is --O-- or --NH--.
In one embodiment, R.sup.10 is aryl
In an exemplary embodiment, R.sup.10 is -phenyl.
In an exemplary embodiment, when Z is --O--, R.sup.11 is --H,
methyl or t-butyl.
In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
Illustrative Compounds of Formula (Ib), each of which may be used
as drug moieties (D) in ADC, include compounds having the following
structures:
##STR00011## ##STR00012## and pharmaceutically acceptable salts or
solvates thereof. The Compounds of Formula (Ic)
In another aspect, the invention provides antibody-drug conjugate
compounds (ADC) having Formula Ic:
AbA.sub.a-W.sub.w--Y.sub.y-D).sub.p Ic comprising an antibody
covalently attached to one or more drug units (moieites). The
antibody-drug conjugate compounds include pharmaceutically
acceptable salts or solvates thereof.
Formula Ic compounds are defined wherein:
Ab is an antibody which binds to one or more tumor-associated
antigen receptors (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank
accession no. NM.sub.--001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.--003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate,
Genbank accession no. NM.sub.--012449);
(4) 0772P(CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424);
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763);
(11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no.
AF455138);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor
potential cation channel, subfamily M, member 4, Genbank accession
no. NM.sub.--017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived
growth factor, Genbank accession no. NP.sub.--003203 or
NM.sub.--003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr
virus receptor) or Hs.73792 Genbank accession no. M26004);
(15) CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated
beta), B29, Genbank accession no. NM.sub.--000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase
anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no.
NM.sub.--030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20R.alpha. (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM.sub.--004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763;
(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3,
BR3, NP.sub.--443177.1);
(27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1);
(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation, Genbank
accession No. NP.sub.--001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled
receptor that is activated by the CXCL13 chemokine, functions in
lymphocyte migration and humoral defense, plays a role in HIV-2
infection and perhaps development of AIDS, lymphoma, myeloma, and
leukemia, Genbank accession No. NP.sub.--001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)
that binds peptides and presents them to CD4+ T lymphocytes,
Genbank accession No. NP.sub.--002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an
ion channel gated by extracellular ATP, may be involved in synaptic
transmission and neurogenesis, deficiency may contribute to the
pathophysiology of idiopathic detrusor instability, Genbank
accession No. NP.sub.--002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank
accession No. NP.sub.--001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis, Genbank accession No. NP.sub.--005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the
immunoglobulin Fc domain that contains C2 type Ig-like and ITAM
domains, may have a role in B-lymphocyte differentiation, Genbank
accession No. NP.sub.--443170.1); and
(35) IRTA2 (Immunoglobulin superfamily receptor translocation
associated 2, a putative immunoreceptor with possible roles in B
cell development and lymphomagenesis; deregulation of the gene by
translocation occurs in some B cell malignancies, Genbank accession
No. NP.sub.--112571.1).
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 8, and
D is a Drug moiety selected from Formulas D.sub.E and D.sub.F:
##STR00013## wherein the wavy line of D.sub.E and D.sub.F indicates
the covalent attachment site to A, W, or Y, and independently at
each location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH;
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
n is an integer ranging from 0 to 6.
In one embodiment --Ww- is -Val-Cit-.
In another embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl.
In yet another embodiment, R.sup.2 and R.sup.6 are each methyl, and
R.sup.9 is --H.
In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
In an exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H, R.sup.7 is
sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3, and R.sup.9
is --H.
In one embodiment, Z is --O-- or --NH--.
In one embodiment, R.sup.10 is aryl.
In an exemplary embodiment, R.sup.10 is -phenyl.
In an exemplary embodiment, when Z is --O--, R.sup.11 is --H,
methyl or t-butyl.
In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H
Exemplary embodiments of Formula Ic ADC have the following
structures:
##STR00014## wherein Ab is an antibody which binds to one or more
tumor-associated antigen receptors (1)-(35); Val is valine; and Cit
is citrulline.
The drug loading is represented by p, the average number of drugs
per antibody in a molecule of Formula I. Drug loading may range
from 1 to 20 drugs (D) per antibody (Ab or mAb). Compositions of
ADC of Formula I include collections of antibodies conjugated with
a range of drugs, from 1 to 20. The average number of drugs per
antibody in preparations of ADC from conjugation reactions may be
characterized by conventional means such as UV/visible
spectroscopy, mass spectrometry, ELISA assay, and HPLC. The
quantitative distribution of ADC in terms of p may also be
determined. In some instances, separation, purification, and
characterization of homogeneous ADC where p is a certain value from
ADC with other drug loadings may be achieved by means such as
reverse phase HPLC or electrophoresis.
For some antibody drug conjugates, p may be limited by the number
of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached.
Typically, fewer than the theoretical maximum of drug moieties are
conjugated to an antibody during a conjugation reaction. An
antibody may contain, for example, many lysine residues that do not
react with the drug-linker intermediate or linker reagent. Only the
most reactive lysine groups may react with an amine-reactive linker
reagent. Generally, antibodies do not contain many, if any, free
and reactive cysteine thiol groups which may be linked to a drug
moiety. Most cysteine thiol residues in the antibodies of the
compounds of the invention exist as disulfide bridges and must be
reduced with a reducing agent such as dithiothreitol (DTT).
Additionally, the antibody must be subjected to denaturing
conditions to reveal reactive nucleophilic groups such as lysine or
cysteine. The loading (drug/antibody ratio) of an ADC may be
controlled in several different manners, including: (i) limiting
the molar excess of drug-linker intermediate or linker reagent
relative to antibody, (ii) limiting the conjugation reaction time
or temperature, and (iii) partial or limiting reductive conditions
for cysteine thiol modification.
It is to be understood that where more than one nucleophilic group
reacts with a drug-linker intermediate, or linker reagent followed
by drug moiety reagent, then the resulting product is a mixture of
ADC compounds with a distribution of one or more drug moieties
attached to an antibody. The average number of drugs per antibody
may be calculated from the mixture by dual ELISA antibody assay,
specific for antibody and specific for the drug. Individual ADC
molecules may be identified in the mixture by mass spectroscopy,
and separated by HPLC, e.g., hydrophobic interaction chromatography
("Effect of drug loading on the pharmacology, pharmacokinetics, and
toxicity of an anti-CD30 antibody-drug conjugate", Hamblett, K. J.,
et al, Abstract No. 624, American Association for Cancer Research;
2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR,
Volume 45, March 2004; "Controlling the Location of Drug Attachment
in Antibody-Drug Conjugates", Alley, S. C., et al, Abstract No.
627, American Association for Cancer Research; 2004 Annual Meeting,
Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004).
Thus, a homogeneous ADC with a single loading value may be isolated
from the conjugation mixture by electrophoresis or
chromatography.
4.3 The Linker Unit
A "Linker unit" (LU) is a bifunctional compound which can be used
to link a Drug unit and an Ligand unit to form Drug-Linker-Ligand
Conjugates, or which are useful in the formation of
immunoconjugates directed against tumor associated antigens. Such
immunoconjugates allow the selective delivery of toxic drugs to
tumor cells.
In one embodiment, the Linker unit of the Drug-Linker Compound and
Drug-Linker-Ligand Conjugate has the formula:
-A.sub.a-W.sub.w--Y.sub.y-- wherein:
-A- is a Stretcher unit;
a is 0 or 1;
each --W-- is independently an Amino Acid unit;
w is independently an integer ranging from 0 to 12;
--Y-- is a Spacer unit; and
y is 0, 1 or 2.
In the Drug-Linker-Ligand Conjugate, the Linker is capable of
linking the Drug moiety and the Ligand unit.
4.3.1 The Stretcher Unit
The Stretcher unit (-A-), when present, is capable of linking a
Ligand unit to an amino acid unit (--W--). In this regard a Ligand
(L) has a functional group that can form a bond with a functional
group of a Stretcher. Useful functional groups that can be present
on a ligand, either naturally or via chemical manipulation include,
but are not limited to, sulfhydryl (--SH), amino, hydroxyl,
carboxy, the anomeric hydroxyl group of a carbohydrate, and
carboxyl. In one aspect, the Ligand functional groups are
sulfhydryl and amino. Sulfhydryl groups can be generated by
reduction of an intramolecular disulfide bond of a Ligand.
Alternatively, sulfhydryl groups can be generated by reaction of an
amino group of a lysine moiety of a Ligand using 2-iminothiolane
(Traut's reagent) or another sulfhydryl generating reagent.
In one embodiment, the Stretcher unit forms a bond with a sulfur
atom of the Ligand unit. The sulfur atom can be derived from a
sulfhydryl group of a Ligand. Representative Stretcher units of
this embodiment are depicted within the square brackets of Formulas
IIIa and IIIb, wherein L-, --W--, --Y--, -D, w and y are as defined
above, and R.sup.17 is selected from --C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 carbocyclo-, --O--(C.sub.1-C.sub.8 alkyl)-,
-arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 carbocyclo)-, --(C.sub.3-C.sub.8
carbocyclo)-C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8
heterocyclo-, --C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8
heterocyclo)-, --(C.sub.3-C.sub.8 heterocyclo)-C.sub.1-C.sub.10
alkylene-, --(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10. It is to be understood from all the exemplary
embodiments of Formula Ia, such as III-VI, that even where not
denoted expressly, from 1 to 20 drug moieties are linked to a
Ligand (p=1-20).
##STR00015##
An illustrative Stretcher unit is that of Formula IIIa wherein
R.sup.17 is --(CH.sub.2).sub.5--:
##STR00016##
Another illustrative Stretcher unit is that of Formula IIIa wherein
R.sup.17 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is
2:
##STR00017##
Still another illustrative Stretcher unit is that of Formula IIIb
wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00018##
In another embodiment, the Stretcher unit is linked to the Ligand
unit via a disulfide bond between a sulfur atom of the Ligand unit
and a sulfur atom of the Stretcher unit. A representative Stretcher
unit of this embodiment is depicted within the square brackets of
Formula IV, wherein R.sup.17, L-, --W--, --Y--, -D, w and y are as
defined above. LS--R.sup.17--C(O)W.sub.w--Y.sub.y-D IV
In yet another embodiment, the reactive group of the Stretcher
contains a reactive site that can form a bond with a primary or
secondary amino group of a Ligand. Example of these reactive sites
include, but are not limited to, activated esters such as
succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters,
tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl
chlorides, isocyanates and isothiocyanates. Representative
Stretcher units of this embodiment are depicted within the square
brackets of Formulas Va and Vb, wherein --R.sup.17--, L-, --W--,
--Y--, -D, w and y are as defined above;
##STR00019##
In yet another aspect, the reactive group of the Stretcher contains
a reactive site that is reactive to a modified carbohydrate's
(--CHO) group that can be present on a Ligand. For example, a
carbohydrate can be mildly oxidized using a reagent such as sodium
periodate and the resulting (--CHO) unit of the oxidized
carbohydrate can be condensed with a Stretcher that contains a
functionality such as a hydrazide, an oxime, a primary or secondary
amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate,
and an arylhydrazide such as those described by Kaneko, T. et al.
(1991) Bioconjugate Chem 2:133-41. Representative Stretcher units
of this embodiment are depicted within the square brackets of
Formulas VIa, VIb, and VIc, wherein --R.sup.17--, L-, --W--, --Y--,
-D, w and y are as defined above.
##STR00020## 4.3.2 The Amino Acid Unit
The Amino Acid unit (--W--), when present, links the Stretcher unit
to the Spacer unit if the Spacer unit is present, links the
Stretcher unit to the Drug moiety if the Spacer unit is absent, and
links the Ligand unit to the Drug unit if the Stretcher unit and
Spacer unit are absent.
W.sub.w-- is a dipeptide, tripeptide, tetrapeptide, pentapeptide,
hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,
undecapeptide or dodecapeptide unit. Each --W-- unit independently
has the formula denoted below in the square brackets, and w is an
integer ranging from 0 to 12:
##STR00021## wherein R.sup.19 is hydrogen, methyl, isopropyl,
isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, --CH.sub.2OH,
--CH(OH)CH.sub.3, --CH.sub.2CH.sub.2SCH.sub.3,
--CH.sub.2CONH.sub.2, --CH.sub.2COOH, --CH.sub.2CH.sub.2CONH.sub.2,
--CH.sub.2CH.sub.2COOH, --(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2,
--(CH.sub.2).sub.3NH.sub.2, --(CH.sub.2).sub.3NHCOCH.sub.3,
--(CH.sub.2).sub.3NHCHO, --(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2,
--(CH.sub.2).sub.4NH.sub.2, --(CH.sub.2).sub.4NHCOCH.sub.3,
--(CH.sub.2).sub.4NHCHO, --(CH.sub.2).sub.3NHCONH.sub.2,
--(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-Pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR00022##
The Amino Acid unit can be enzymatically cleaved by one or more
enzymes, including a tumor-associated protease, to liberate the
Drug unit (-D), which in one embodiment is protonated in vivo upon
release to provide a Drug (D).
Illustrative W.sub.w units are represented by formulas
(VII)-(IX):
TABLE-US-00001 (VII) ##STR00023## wherein R.sup.20 and R.sup.21 are
as follows: R.sup.20 R.sup.21 benzyl (CH.sub.2).sub.4NH.sub.2;
methyl (CH.sub.2).sub.4NH.sub.2; isopropyl
(CH.sub.2).sub.4NH.sub.2; isopropyl (CH.sub.2).sub.3NHCONH.sub.2;
benzyl (CH.sub.2).sub.3NHCONH.sub.2; isobutyl
(CH.sub.2).sub.3NHCONH.sub.2; sec-butyl
(CH.sub.2).sub.3NHCONH.sub.2; ##STR00024##
(CH.sub.2).sub.3NHCONH.sub.2; benzyl methyl; and benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2; (VIII) ##STR00025## wherein
R.sup.20, R.sup.21and R.sup.22 are as follows: R.sup.20 R.sup.21
R.sup.22 benzyl benzyl (CH.sub.2).sub.4NH.sub.2; isopropyl benzyl
(CH.sub.2).sub.4NH.sub.2; and H benzyl (CH.sub.2).sub.4NH.sub.2;
(IX) ##STR00026## wherein R.sup.20, R.sup.21, R.sup.22 and R.sup.23
are as follows: R.sup.20 R.sup.21 R.sup.22 R.sup.23 H benzyl
isobutyl H; and methyl isobutyl methyl isobutyl.
Exemplary Amino Acid units include, but are not limited to, units
of formula (VII) where: R.sup.20 is benzyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.20 isopropyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.20 isopropyl and R.sup.21 is
--(CH.sub.2).sub.3NHCONH.sub.2. Another exemplary Amino Acid unit
is a unit of formula (VIII) wherein R.sup.20 is benzyl, R.sup.21 is
benzyl, and R.sup.22 is --(CH.sub.2).sub.4NH.sub.2.
Useful --W.sub.W-- units can be designed and optimized in their
selectivity for enzymatic cleavage by a particular enzymes, for
example, a tumor-associated protease. In one embodiment, a
--W.sub.w-- unit is that whose cleavage is catalyzed by cathepsin
B, C and D, or a plasmin protease.
In one embodiment, --W.sub.w-- is a dipeptide, tripeptide,
tetrapeptide or pentapeptide.
When R.sup.19, R.sup.20, R.sup.21, R.sup.22, or R.sup.23 is other
than hydrogen, the carbon atom to which R.sup.19, R.sup.20,
R.sup.21, R.sup.22 or R.sup.23 is attached is chiral.
Each carbon atom to which R.sup.19, R.sup.20, R.sup.21, R.sup.22 or
R.sup.23 is attached is independently in the (S) or (R)
configuration.
In one aspect of the Amino Acid unit, the Amino Acid unit is
valine-citrulline. In another aspect, the Amino Acid unit is
phenylalanine-lysine (i.e. fk). In yet another aspect of the Amino
Acid unit, the Amino Acid unit is N-methylvaline-citrulline. In yet
another aspect, the Amino Acid unit is 5-aminovaleric acid, homo
phenylalanine lysine, tetraisoquinolinecarboxylate lysine,
cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine
lysine, glycine serine valine glutamine and isonepecotic acid.
In certain embodiments, the Amino Acid unit can comprise natural
amino acids. In other embodiments, the Amino Acid unit can comprise
non-natural amino acids.
4.3.3 The Spacer Unit
The Spacer unit (--Y--), when present, links an Amino Acid unit to
the Drug moiety when an Amino Acid unit is present. Alternately,
the Spacer unit links the Stretcher unit to the Drug moiety when
the Amino Acid unit is absent. The Spacer unit also links the Drug
moiety to the Ligand unit when both the Amino Acid unit and
Stretcher unit are absent.
Spacer units are of two general types: self-immolative and non
self-immolative. A non self-immolative Spacer unit is one in which
part or all of the Spacer unit remains bound to the Drug moiety
after cleavage, particularly enzymatic, of an Amino Acid unit from
the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound.
Examples of a non self-immolative Spacer unit include, but are not
limited to a (glycine-glycine) Spacer unit and a glycine Spacer
unit (both depicted in FIG. 20) (infra). When an Exemplary Compound
containing a glycine-glycine Spacer unit or a glycine Spacer unit
undergoes enzymatic cleavage via a tumor-cell associated-protease,
a cancer-cell-associated protease or a lymphocyte-associated
protease, a glycine-glycine-Drug moiety or a glycine-Drug moiety is
cleaved from L-A.sub.a-Ww-. In one embodiment, an independent
hydrolysis reaction takes place within the target cell, cleaving
the glycine - - - Drug moiety bond and liberating the Drug.
In another embodiment, --Y.sub.y-- is a p-aminobenzyl alcohol (PAB)
unit (see FIGS. 21 and 22) whose phenylene portion is substituted
with Q.sub.m wherein Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4.
In one embodiment, a non self-immolative Spacer unit (--Y--) is
-Gly-Gly-. In another embodiment, a non self-immolative the Spacer
unit (--Y--) is -Gly-.
In one embodiment, a Drug-Linker Compound or a Drug-Linker Ligand
Conjugate is provided in which the Spacer unit is absent (y=0), or
a pharmaceutically acceptable salt or solvate thereof.
Alternatively, an Exemplary Compound containing a self-immolative
Spacer unit can release -D without the need for a separate
hydrolysis step. In this embodiment, --Y-- is a PAB group that is
linked to --W.sub.w-- via the amino nitrogen atom of the PAB group,
and connected directly to -D via a carbonate, carbamate or ether
group. Without being bound by any particular theory or mechanism,
FIG. 21 depicts a possible mechanism of Drug release of a PAB group
which is attached directly to -D via a carbamate or carbonate group
espoused by Toki et al. (2002) J. Org. Chem. 67:1866-1872.
In FIG. 21 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; m is an integer ranging from
0-4; and p ranges from 1 to about 20.
Without being bound by any particular theory or mechanism, FIG. 22
depicts a possible mechanism of Drug release of a PAB group which
is attached directly to -D via an ether or amine linkage.
In FIG. 22 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; m is an integer ranging from
0-4; and p ranges from 1 to about 20.
Other examples of self-immolative spacers include, but are not
limited to, aromatic compounds that are electronically similar to
the PAB group such as 2-aminoimidazol-5-methanol derivatives (Hay
et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho or
para-aminobenzylacetals. Spacers can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al.,
Chemistry Biology, 1995, 2, 223), appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J.
Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid
amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867).
Elimination of amine-containing drugs that are substituted at the
a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27,
1447) are also examples of self-immolative spacer useful in
Exemplary Compounds.
In one embodiment, the Spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit as depicted in FIG. 23, which
can be used to incorporate and release multiple drugs.
In FIG. 23 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; m is an integer ranging from
0-4; n is 0 or 1; and p ranges raging from 1 to about 20.
In one embodiment, the -D moieties are the same. In yet another
embodiment, the -D moieties are different.
In one aspect, Spacer units (--Y.sub.y--) are represented by
Formulas (X)-(XII):
##STR00027## wherein Q is --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -halogen, -nitro or -cyano; and m is
an integer ranging from 0-4;
##STR00028##
Embodiments of the Formula Ia' and Ic antibody-drug conjugate
compounds include:
##STR00029## wherein w and y are each 0,
##STR00030##
4.4 The Drug Unit
Moiety
The drug moiety (D) of the antibody drug conjugates (ADC) are of
the dolastatin/auristatin type (U.S. Pat. Nos. 5,635,483;
5,780,588) which have been shown to interfere with microtubule
dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke
et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)
and have anticancer (U.S. Pat. No. 5,663,149) and antifungal
activity (Pettit et al. (1998) Antimicrob. Agents Chemother.
42:2961-2965)
D is a Drug unit (moiety) having a nitrogen atom that can form a
bond with the Spacer unit when y=1 or 2, with the C-terminal
carboxyl group of an Amino Acid unit when y=0, with the carboxyl
group of a Stretcher unit when w and y=0, and with the carboxyl
group of a Drug unit when a, w, and y=0. It is to be understood
that the terms "drug unit" and "drug moiety" are synonymous and
used interchangeably herein.
In one embodiment, -D is either formula D.sub.E or D.sub.F:
##STR00031## wherein, independently at each location:
R.sup.2 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.4 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
R.sup.5 is selected from H and methyl;
or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and have the
formula --(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6;
R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle);
each R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8
alkyl);
R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl;
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is C.sub.1-C.sub.8
alkyl;
R.sup.11 is selected from H, C.sub.1-C.sub.20 alkyl, aryl,
C.sub.3-C.sub.8 heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
m is an integer ranging from 1-1000;
R.sup.13 is C.sub.2-C.sub.8 alkyl;
R.sup.14 is H or C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl;
each occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8
alkyl, or --(CH.sub.2).sub.n--COOH;
R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and
n is an integer ranging from 0 to 6.
In one embodiment, R.sup.3, R.sup.4 and R.sup.7 are independently
isopropyl or sec-butyl and R.sup.5 is --H. In an exemplary
embodiment, R.sup.3 and R.sup.4 are each isopropyl, R.sup.5 is H,
and R.sup.7 is sec-butyl.
In another embodiment, R.sup.2 and R.sup.6 are each methyl, and
R.sup.9 is H.
In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
In an exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.2 and R.sup.6 are each methyl, R.sup.5 is H, R.sup.7 is
sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3, and R.sup.9
is H.
In one embodiment, Z is --O-- or --NH--.
In one embodiment, R.sup.10 is aryl
In an exemplary embodiment, R.sup.10 is -phenyl.
In an exemplary embodiment, when Z is --O--, R.sup.11 is H, methyl
or t-butyl.
In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH.
In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
Illustrative Drug units (-D) include the drug units having the
following structures:
##STR00032## ##STR00033## and pharmaceutically acceptable salts or
solvates thereof.
In one aspect, hydrophilic groups, such as but not limited to
triethylene glycol esters (TEG), as shown above, can be attached to
the Drug Unit at R.sup.11. Without being bound by theory, the
hydrophilic groups assist in the internalization and
non-agglomeration of the Drug Unit.
4.5 The Ligand Unit
The Ligand unit (L-) includes within its scope any unit of a Ligand
(L) that binds or reactively associates or complexes with a
receptor, antigen or other receptive moiety associated with a given
target-cell population. A Ligand is a molecule that binds to,
complexes with, or reacts with a moiety of a cell population sought
to be therapeutically or otherwise biologically modified. In one
aspect, the Ligand unit acts to deliver the Drug unit to the
particular target cell population with which the Ligand unit
reacts. Such Ligands include, but are not limited to, large
molecular weight proteins such as, for example, full-length
antibodies, antibody fragments, smaller molecular weight proteins,
polypeptide or peptides, lectins, glycoproteins, non-peptides,
vitamins, nutrient-transport molecules (such as, but not limited
to, transferrin), or any other cell binding molecule or
substance.
A Ligand unit can form a bond to a Stretcher unit, an Amino Acid
unit, a Spacer Unit, or a Drug Unit. A Ligand unit can form a bond
to a Linker unit via a heteroatom of the Ligand. Heteroatoms that
may be present on a Ligand unit include sulfur (in one embodiment,
from a sulfhydryl group of a Ligand), oxygen (in one embodiment,
from a carbonyl, carboxyl or hydroxyl group of a Ligand) and
nitrogen (in one embodiment, from a primary or secondary amino
group of a Ligand). These heteroatoms can be present on the Ligand
in the Ligand's natural state, for example a naturally-occurring
antibody, or can be introduced into the Ligand via chemical
modification.
In one embodiment, a Ligand has a sulthydryl group and the Ligand
bonds to the Linker unit via the sulthydryl group's sulfur
atom.
In yet another aspect, the Ligand has one or more lysine residues
that can be chemically modified to introduce one or more sulfhydryl
groups. The Ligand unit bonds to the Linker unit via the sulfhydryl
group's sulfur atom. The reagents that can be used to modify
lysines include, but are not limited to, N-succinimidyl
S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride
(Traut's Reagent).
In another embodiment, the Ligand can have one or more carbohydrate
groups that can be chemically modified to have one or more
sulthydryl groups. The Ligand unit bonds to the Linker Unit, such
as the Stretcher Unit, via the sulfhydryl group's sulfur atom.
In yet another embodiment, the Ligand can have one or more
carbohydrate groups that can be oxidized to provide an aldehyde
(--CHO) group (see, for e.g., Laguzza, et al., J. Med. Chem. 1989,
32(3), 548-55). The corresponding aldehyde can form a bond with a
Reactive Site on a Stretcher. Reactive sites on a Stretcher that
can react with a carbonyl group on a Ligand include, but are not
limited to, hydrazine and hydroxylamine. Other protocols for the
modification of proteins for the attachment or association of Drug
Units are described in Coligan et al., Current Protocols in Protein
Science, vol. 2, John Wiley & Sons (2002), incorporated herein
by reference.
Useful non-immunoreactive protein, polypeptide, or peptide Ligands
include, but are not limited to, transferrin, epidermal growth
factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide,
platelet-derived growth factor, IL-2, IL-6, transforming growth
factors ("TGF"), such as TGF-.alpha. and TGF-.beta., vaccinia
growth factor ("VGF"), insulin and insulin-like growth factors I
and II, lectins and apoprotein from low density lipoprotein.
Useful polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of immunized animals.
Various procedures well known in the art may be used for the
production of polyclonal antibodies to an antigen-of-interest. For
example, for the production of polyclonal antibodies, various host
animals can be immunized by injection with an antigen of interest
or derivative thereof, including but not limited to rabbits, mice,
rats, and guinea pigs. Various adjuvants may be used to increase
the immunological response, depending on the host species, and
including but not limited to Freund's (complete and incomplete)
adjuvant, mineral gels such as aluminum hydroxide, surface active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol,
and potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art.
Useful monoclonal antibodies are homogeneous populations of
antibodies to a particular antigenic determinant (e.g., a cancer
cell antigen, a viral antigen, a microbial antigen, a protein, a
peptide, a carbohydrate, a chemical, nucleic acid, or fragments
thereof). A monoclonal antibody (mAb) to an antigen-of-interest can
be prepared by using any technique known in the art which provides
for the production of antibody molecules by continuous cell lines
in culture. These include, but are not limited to, the hybridoma
technique originally described by Kohler and Milstein (1975, Nature
256, 495-497), the human B cell hybridoma technique (Kozbor et al.,
1983, Immunology Today 4: 72), and the EBV-hybridoma technique
(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, Inc., pp. 77-96). Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any
subclass thereof. The hybridoma producing the mAbs of use in this
invention may be cultivated in vitro or in vivo.
Useful monoclonal antibodies include, but are not limited to, human
monoclonal antibodies, humanized monoclonal antibodies, antibody
fragments, or chimeric human-mouse (or other species) monoclonal
antibodies. Human monoclonal antibodies may be made by any of
numerous techniques known in the art (e.g., Teng et al., 1983,
Proc. Natl. Acad. Sci. USA. 80, 7308-7312; Kozbor et al., 1983,
Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol.
92, 3-16).
The antibody can also be a bispecific antibody. Methods for making
bispecific antibodies are known in the art. Traditional production
of full-length bispecific antibodies is based on the coexpression
of two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (Milstein et al., 1983, Nature
305:537-539). Because of the random assortment of immunoglobulin
heavy and light chains, these hybridomas (quadromas) produce a
potential mixture of 10 different antibody molecules, of which only
one has the correct bispecific structure. Similar procedures are
disclosed in International Publication No. WO 93/08829, and in
Traunecker et al., EMBO J. 10:3655-3659 (1991).
According to a different approach, antibody variable domains with
the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. It is preferred to have the first heavy-chain
constant region (C.sub.H1) containing the site necessary for light
chain binding, present in at least one of the fusions. Nucleic
acids with sequences encoding the immunoglobulin heavy chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. This provides for great flexibility
in adjusting the mutual proportions of the three polypeptide
fragments in embodiments when unequal ratios of the three
polypeptide chains used in the construction provide the optimum
yields. It is, however, possible to insert the coding sequences for
two or all three polypeptide chains in one expression vector when
the expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
In an embodiment of this approach, the bispecific antibodies have a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure facilitates the separation of the desired
bispecific compound from unwanted immunoglobulin chain
combinations, as the presence of an immunoglobulin light chain in
only one half of the bispecific molecule provides for a facile way
of separation (International Publication No. WO 94/04690) which is
incorporated herein by reference in its entirety.
For further details for generating bispecific antibodies see, for
example, Suresh et al., Methods in Enzymology, 1986, 121:210;
Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et
al., 1992, Bio/Technology 10:163-167; Carter et al., 1995, J. of
Hematotherapy 4:463-470; Merchant et al., 1998, Nature
Biotechnology 16:677-681. Using such techniques, bispecific
antibodies can be prepared for use in the treatment or prevention
of disease as defined herein.
Bifunctional antibodies are also described, in European Patent
Publication No. EPA 0 105 360. As disclosed in this reference,
hybrid or bifunctional antibodies can be derived either
biologically, i.e., by cell fusion techniques, or chemically,
especially with cross-linking agents or disulfide-bridge forming
reagents, and may comprise whole antibodies or fragments thereof.
Methods for obtaining such hybrid antibodies are disclosed for
example, in International Publication WO 83/03679, and European
Patent Publication No. EPA 0 217 577, both of which are
incorporated herein by reference.
The antibody can be a functionally active fragment, derivative or
analog of an antibody that immunospecifically binds to cancer cell
antigens, viral antigens, or microbial antigens or other antibodies
bound to tumor cells or matrix. In this regard, "functionally
active" means that the fragment, derivative or analog is able to
elicit anti-anti-idiotype antibodies that recognize the same
antigen that the antibody from which the fragment, derivative or
analog is derived recognized. Specifically, in an exemplary
embodiment the antigenicity of the idiotype of the immunoglobulin
molecule can be enhanced by deletion of framework and CDR sequences
that are C-terminal to the CDR sequence that specifically
recognizes the antigen. To determine which CDR sequences bind the
antigen, synthetic peptides containing the CDR sequences can be
used in binding assays with the antigen by any binding assay method
known in the art (e.g., the BIA core assay) (See, for e.g., Kabat
et al., 1991, Sequences of Proteins of Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E
et al., 1980, J. of Immunology 125(3):961-969).
Other useful antibodies include fragments of antibodies such as,
but not limited to, F(ab').sub.2 fragments, which contain the
variable region, the light chain constant region and the CH1 domain
of the heavy chain can be produced by pepsin digestion of the
antibody molecule, and Fab fragments, which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragments. Other
useful antibodies are heavy chain and light chain dimers of
antibodies, or any minimal fragment thereof such as Fvs or single
chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.
4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-54), or any other molecule with the same specificity
as the antibody.
Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are useful antibodies. A chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal and human immunoglobulin constant regions.
(See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et
al., U.S. Pat. No. 4,816,397, which are incorporated herein by
reference in their entirety.) Humanized antibodies are antibody
molecules from non-human species having one or more complementarity
determining regions (CDRs) from the non-human species and a
framework region from a human immunoglobulin molecule. (See, e.g.,
Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by
reference in its entirety.) Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in International
Publication No. WO 87/02671; European Patent Publication No.
184,187; European Patent Publication No. 171496; European Patent
Publication No. 173494; International Publication No. WO 86/01533;
U.S. Pat. No. 4,816,567; European Patent Publication No. 12,023;
Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc.
Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.
139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA
84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood
et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl.
Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207;
Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539;
Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al., 1988, J. Immunol.
141:4053-4060; each of which is incorporated herein by reference in
its entirety.
Completely human antibodies are particularly desirable and can be
produced using transgenic mice that are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of a polypeptide of the invention. Monoclonal
antibodies directed against the antigen can be obtained using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which is
incorporated herein by reference in its entirety. Other human
antibodies can be obtained commercially from, for example, Abgenix,
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).
Completely human antibodies that recognize a selected epitope can
be generated using a technique referred to as "guided selection."
In this approach a selected non-human monoclonal antibody, e.g., a
mouse antibody, is used to guide the selection of a completely
human antibody recognizing the same epitope. (Jespers et al. (1994)
Biotechnology 12:899-903). Human antibodies can also be produced
using various techniques known in the art, including phage display
libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991); Quan, M. P. and
Carter, P. 2002. The rise of monoclonal antibodies as therapeutics.
In Anti-IgE and Allergic Disease, Jardieu, P. M. and Fick Jr., R.
B, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp.
427-469).
In other embodiments, the antibody is a fusion protein of an
antibody, or a functionally active fragment thereof, for example in
which the antibody is fused via a covalent bond (e.g., a peptide
bond), at either the N-terminus or the C-terminus to an amino acid
sequence of another protein (or portion thereof, preferably at
least 10, 20 or 50 amino acid portion of the protein) that is not
the antibody. Preferably, the antibody or fragment thereof is
covalently linked to the other protein at the N-terminus of the
constant domain.
Antibodies include analogs and derivatives that are either
modified, i.e., by the covalent attachment of any type of molecule
as long as such covalent attachment permits the antibody to retain
its antigen binding immunospecificity. For example, but not by way
of limitation, the derivatives and analogs of the antibodies
include those that have been further modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular antibody unit or other protein,
etc. Any of numerous chemical modifications can be carried out by
known techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis in the
presence of tunicamycin, etc. Additionally, the analog or
derivative can contain one or more unnatural amino acids.
The antibodies include antibodies having modifications (e.g.,
substitutions, deletions or additions) in amino acid residues that
interact with Fc receptors. In particular, antibodies include
antibodies having modifications in amino acid residues identified
as involved in the interaction between the anti-Fc domain and the
FcRn receptor (see, e.g., International Publication No. WO
97/34631, which is incorporated herein by reference in its
entirety). Antibodies immunospecific for a cancer cell antigen can
be obtained commercially, for example, from Genentech (San
Francisco, Calif.) or produced by any method known to one of skill
in the art such as, e.g., chemical synthesis or recombinant
expression techniques. The nucleotide sequence encoding antibodies
immunospecific for a cancer cell antigen can be obtained, e.g.,
from the GenBank database or a database like it, the literature
publications, or by routine cloning and sequencing.
In a specific embodiment, known antibodies for the treatment or
prevention of cancer can be used. Antibodies immunospecific for a
cancer cell antigen can be obtained commercially or produced by any
method known to one of skill in the art such as, e.g., recombinant
expression techniques. The nucleotide sequence encoding antibodies
immunospecific for a cancer cell antigen can be obtained, e.g.,
from the GenBank database or a database like it, the literature
publications, or by routine cloning and sequencing. Examples of
antibodies available for the treatment of cancer include, but are
not limited to, humanized anti-HER2 monoclonal antibody,
HERCEPTIN.RTM. (trastuzumab; Genentech) for the treatment of
patients with metastatic breast cancer; RITUXAN.RTM. (rituximab;
Genentech) which is a chimeric anti-CD20 monoclonal antibody for
the treatment of patients with non-Hodgkin's lymphoma; OvaRex
(AltaRex Corporation, MA) which is a murine antibody for the
treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is
a murine IgG.sub.2a antibody for the treatment of colorectal
cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) which is an
anti-EGFR IgG chimeric antibody for the treatment of epidermal
growth factor positive cancers, such as head and neck cancer;
Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the
treatment of sarcoma; Campath I/H (Leukosite, Mass.) which is a
humanized IgG.sub.1 antibody for the treatment of chronic
lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc.,
CA) which is a humanized anti-CD33 IgG antibody for the treatment
of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc.,
NJ) which is a humanized anti-CD22 IgG antibody for the treatment
of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc.,
CA) which is a humanized anti-HLA-DR antibody for the treatment of
non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a
radiolabeled murine anti-HLA-Dr10 antibody for the treatment of
non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a
humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or
non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA) which is an
anti-VEGF humanized antibody for the treatment of lung and
colorectal cancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen,
Calif.) which is an anti-CD22 antibody for the treatment of
non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ) which is a
humanized anti-CEA antibody for the treatment of colorectal
cancer.
Other antibodies useful in the treatment of cancer include, but are
not limited to, antibodies against the following antigens: CA125
(ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6
(carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha
fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline
phosphatase (carcinomas), prostate specific antigen (prostate),
prostatic acid phosphatase (prostate), epidermal growth factor
(carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3
(carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor
(carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA
(colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate),
IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's
lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human
chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40
(lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma),
and Neu oncogene product (carcinomas). Some specific, useful
antibodies include, but are not limited to, BR96 mAb (Trail, P. A.,
Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J.,
Casazza, A. M., Firestone, R. A., Hellstrom, I., Hellstrom, K. E.,
"Cure of Xenografted Human Carcinomas by BR96-Doxorubicin
Immunoconjugates" Science 1993, 261, 212-215), BR64 (Trail, P A,
Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M.
E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M.,
Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R.
S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B.,
Hellstrom, K. E., and Hellstrom, I. "Effect of Linker Variation on
the Stability, Potency, and Efficacy of Carcinoma-reactive
BR64-Doxorubicin Immunoconjugates" Cancer Research 1997, 57,
100-105, mAbs against the CD40 antigen, such as S2C6 mAb
(Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and
Wahl, A. F. "Agonistic properties and in vivo antitumor activity of
the anti-CD-40 antibody, SGN-14" Cancer Res. 2000, 60, 3225-3231),
mAbs against the CD70 antigen, such as 1F6 mAb and 2F2 mAb, and
mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen,
K. J., Cheng, L., Avila, D., and Podack, E. R. "Functional effects
of CD30 on a large granular lymphoma cell line YT" J. Immunol.,
151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res.
62(13):3736-42). Many other internalizing antibodies that bind to
tumor associated antigens can be used and have been reviewed
(Franke, A. E., Sievers, E. L., and Scheinberg, D. A., "Cell
surface receptor-targeted therapy of acute myeloid leukemia: a
review" Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L.,
"Monoclonal antibody treatment of solid tumors: a coming of age"
Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S.,
Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
In certain embodiments, the antibody is not Trastuzumab (full
length, humanized anti-HER2 (MW 145167)), HerceptinF(ab').sub.2
(derived from anti-HER2 enzymatically (MW 100000)), 4D5
(full-length, murine antiHER2, from hybridoma), rhu4D5 (transiently
expressed, full-length humanized antibody), rhuFab4D5 (recombinant
humanized Fab (MW 47738)), 4D5Fc8 (full-length, murine antiHER2,
with mutated FcRn binding domain), or Hg ("Hingeless" full-length
humanized 4D5, with heavy chain hinge cysteines mutated to serines.
Expressed in E. coli (therefore non-glycosylated)).
In another specific embodiment, known antibodies for the treatment
or prevention of an autoimmune disease are used in accordance with
the compositions and methods of the invention. Antibodies
immunospecific for an antigen of a cell that is responsible for
producing autoimmune antibodies can be obtained from any
organization (e.g., a university scientist or a company) or
produced by any method known to one of skill in the art such as,
e.g., chemical synthesis or recombinant expression techniques. In
another embodiment, useful antibodies are immunospecific for the
treatment of autoimmune diseases include, but are not limited to,
Anti-Nuclear Antibody; Anti-ds DNA; Anti-ss DNA, Anti-Cardiolipin
Antibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SM
Antibody; Anti-Mitochondrial Antibody; Thyroid Antibody; Microsomal
Antibody; Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo;
Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital
Cells Antibody; Anti-Histones; Anti-RNP; C-ANCA; P-ANCA; Anti
centromere; Anti-Fibrillarin, and Anti-GBM Antibody.
In certain embodiments, useful antibodies can bind to both a
receptor or a receptor complex expressed on an activated
lymphocyte. The receptor or receptor complex can comprise an
immunoglobulin gene superfamily member, a TNF receptor superfamily
member, an integrin, a cytokine receptor, a chemokine receptor, a
major histocompatibility protein, a lectin, or a complement control
protein. Non-limiting examples of suitable immunoglobulin
superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79,
CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of
suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas,
CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,
osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and
APO-3. Non-limiting examples of suitable integrins are CD11a,
CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e,
CD49f, CD103, and CD104. Non-limiting examples of suitable lectins
are C-type, S-type, and I-type lectin.
In one embodiment, the Ligand binds to an activated lymphocyte that
is associated with an autoimmune disease.
In another specific embodiment, useful Ligands immunospecific for a
viral or a microbial antigen are monoclonal antibodies. The
antibodies may be chimeric, humanized or human monoclonal
antibodies. As used herein, the term "viral antigen" includes, but
is not limited to, any viral peptide, polypeptide protein (e.g.,
HIV gp120, HIV nef, RSV F glycoprotein, influenza virus
neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes
simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis
B surface antigen) that is capable of eliciting an immune response.
As used herein, the term "microbial antigen" includes, but is not
limited to, any microbial peptide, polypeptide, protein,
saccharide, polysaccharide, or lipid molecule (e.g., a bacterial,
fungi, pathogenic protozoa, or yeast polypeptide including, e.g.,
LPS and capsular polysaccharide 5/8) that is capable of eliciting
an immune response.
Antibodies immunospecific for a viral or microbial antigen can be
obtained commercially, for example, from BD Biosciences (San
Francisco, Calif.), Chemicon International, Inc. (Temecula,
Calif.), or Vector Laboratories, Inc. (Burlingame, Calif.) or
produced by any method known to one of skill in the art such as,
e.g., chemical synthesis or recombinant expression techniques. The
nucleotide sequence encoding antibodies that are immunospecific for
a viral or microbial antigen can be obtained, e.g., from the
GenBank database or a database like it, literature publications, or
by routine cloning and sequencing.
In a specific embodiment, useful Ligands are those that are useful
for the treatment or prevention of viral or microbial infection in
accordance with the methods disclosed herein. Examples of
antibodies available useful for the treatment of viral infection or
microbial infection include, but are not limited to, SYNAGIS
(MedImmune, Inc., MD) which is a humanized anti-respiratory
syncytial virus (RSV) monoclonal antibody useful for the treatment
of patients with RSV infection; PRO542 (Progenies) which is a CD4
fusion antibody useful for the treatment of HIV infection; OSTAVIR
(Protein Design Labs, Inc., CA) which is a human antibody useful
for the treatment of hepatitis B virus; PROTOVIR (Protein Design
Labs, Inc., CA) which is a humanized IgG.sub.1 antibody useful for
the treatment of cytomegalovirus (CMV); and anti-LPS
antibodies.
Other antibodies useful in the treatment of infectious diseases
include, but are not limited to, antibodies against the antigens
from pathogenic strains of bacteria (Streptococcus pyogenes,
Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria
meningitidis, Corynebacterium diphtheriae, Clostridium botulinum,
Clostridium perfringens, Clostridium tetani, Hemophilus influenzae,
Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella
rhinoscleromotis, Staphylococc aureus, Vibrio colerae, Escherichia
coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus,
Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia
enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,
Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Salmonella typhimurium, Treponema pallidum, Treponema pertenue,
Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,
Leptospira icterohemorrhagiae, Mycobacterium tuberculosis,
Pneumocystis carinii, Francisella tularensis, Brucella abortus,
Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia
prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic
fungi (Coccidioides immitis, Aspergillus fumigatus, Candida
albicans, Blastomyces dermatitidis, Cryptococcus neoformans,
Histoplasma capsulatum); protozoa (Entomoeba histolytica,
Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis,
Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma
rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania
tropica, Leishmania braziliensis, Pneumocystis pneumonia,
Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or
Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris
lumbricoides, Trichinella spiralis, Strongyloides stercoralis,
Schistosoma japonicum, Schistosoma mansoni, Schistosoma
haematobium, and hookworms).
Other antibodies useful in this invention for treatment of viral
disease include, but are not limited to, antibodies against
antigens of pathogenic viruses, including as examples and not by
limitation: Poxyiridae, Herpesviridae, Herpes Simplex virus 1,
Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae,
Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza
viruses, parainfluenza viruses, mumps, measles, respiratory
syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,
Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis
E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae,
Rotoviridae, and Human Immunodeficiency Virus.
In attempts to discover effective cellular targets for cancer
diagnosis and therapy, researchers have sought to identify
transmembrane or otherwise tumor-associated polypeptides that are
specifically expressed on the surface of one or more particular
type(s) of cancer cell as compared to on one or more normal
non-cancerous cell(s). Often, such tumor-associated polypeptides
are more abundantly expressed on the surface of the cancer cells as
compared to on the surface of the non-cancerous cells. The
identification of such tumor-associated cell surface antigen
polypeptides has given rise to the ability to specifically target
cancer cells for destruction via antibody-based therapies.
Antibodies which comprise Ab in Formula Ic antibody drug conjugates
(ADC) and which may be useful in the treatment of cancer include,
but are not limited to, antibodies against tumor-associated
antigens (TAA). Such tumor-associated antigens are known in the
art, and can prepared for use in generating antibodies using
methods and information which are well known in the art. Examples
of TAA include (1)-(35), but are not limited to TAA (1)-(35) listed
below. For convenience, information relating to these antigens, all
of which are known in the art, is listed below and includes names,
alternative names, Genbank accession numbers and primary
reference(s). Tumor-associated antigens targeted by antibodies
include all amino acid sequence variants and isoforms possessing at
least about 70%, 80%, 85%, 90%, or 95% sequence identity relative
to the sequences identified in the corresponding sequences listed
(SEQ ID NOS: 1-35) or the sequences identified in the cited
references. In some embodiments, TAA having amino acid sequence
variants exhibit substantially the same biological properties or
characteristics as a TAA having the sequence found in the
corresponding sequences listed (SEQ ID NOS: 1-35). For example, a
TAA having a variant sequence generally is able to bind
specifically to an antibody that binds specifically to the TAA with
the corresponding sequence listed. The sequences and disclosure
specifically recited herein are expressly incorporated by
reference.
Tumor-Associated Antigens (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank
accession no. NM.sub.--001203, ten Dijke, P., et al. Science 264
(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997));
WO2004063362 (Claim 2); WO2003042661 (Claim 12); US2003134790-A1
(Page 38-39); WO2002102235 (Claim 13; Page 296); WO2003055443 (Page
91-92); WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim
6); WO2003024392 (Claim 2; FIG. 112); WO200298358 (Claim 1; Page
183); WO200254940 (Page 100-101); WO200259377 (Page 349-350);
WO200230268 (Claim 27; Page 376); WO200148204 (Example; FIG. 4)
NP.sub.--001194 bone morphogenetic protein receptor, type
IB/pid=NP.sub.--001194.1--Cross-references: MIM:603248;
NP.sub.--001194.1; NM.sub.--001203.sub.--1 502 aa
TABLE-US-00002 (SEQ ID NO: 1)
MLLRSAGKLNVGTKKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTDGYCFTMIEED
DSGLPVVTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERNECNKDLHPTLPPLKNRDFVD
GPIHHRALLISVTVCSLLLVLIILFCYFRYKRQETRPRYSIGLEQDETYIPPGESLRDLI
EQSQSSGSGSGLPLLVQRTIAKQIQMVKQIGKGRYGEVWMGKWRGEKVAVKVFFTTEEAS
WFRETEIYQTVLMRHENILGFIAADIKGTGSWTQLYLITDYHENGSLYDYLKSTTLDAKS
MLKLAYSSVSGLCHLHTEIFSTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVKFISD
TNEVDIPPNTRVGTKRYMPPEVLDESLNRNHFQSYIMADMYSFGLILWEVARRCVSGGIV
EEYQLPYHDLVPSDPSYEDMREIVCIKKLRPSFPNRWSSDECLRQMGKLMTECWAHNPAS
RLTALRVKKTLAKMSESQDIKL
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.--003486);
Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395
(6699):288-291 (1998), Gaugitsch, H. W., et al. (1992) J. Biol.
Chem. 267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842
(Example IV); WO2003042661 (Claim 12); WO2003016475 (Claim 1);
WO200278524 (Example 2); WO200299074 (Claim 19; Page 127-129);
WO200286443 (Claim 27; Pages 222, 393); WO2003003906 (Claim 10;
Page 293); WO200264798 (Claim 33; Page 93-95); WO200014228 (Claim
5; Page 133-136); US2003224454 (FIG. 3); WO2003025138 (Claim 12;
Page 150); NP.sub.--003477 solute carrier family 7 (cationic amino
acid transporter, y+ system), member 5/pid=NP.sub.--003477.3--Homo
sapiens Cross-references: MIM:600182; NP.sub.--003477.3;
NM.sub.--015923; NM.sub.--003486.sub.--1 507 aa
TABLE-US-00003 (SEQ ID NO: 2)
MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNITLLNGVAIIV
GTIIGSGIFVTPTGVLKEAGSPGLALVVWAACGVFSIVGALCYAELGTTISKSGGDYAYM
LEVYGSLPAFLKLWIELLIIRPSSQYIVALVFATYLLKPLFPTCPVPEEAAKLVACLCVL
LLTAVNCYSVKAATRVQDAFAAAKLLALALIILLGFVQIGKGVVSNLDPNFSFEGTKLDV
GNIVLALYSGLFAYGGWNYLNFVTEEMINPYRNLPLAIIISLPIVTLVYVLTNLAYFTTL
STEQMLSSEAVAVDFGNYHLGVMSWIIPVFVGLSCFGSVNGSLFTSSRLFFVGSREGHLP
SILSMIHPQLLTPVPSLVFTCVMTLLYAFSKDIFSVINFFSFFNWLCVALAIIGMIWLRH
RKPELERPIKVNLALPVFFILACLFLIAVSFWKTPVECGIGFTIILSGLPVYFFGVWWKN
KPKWLLQGIFSTTVLCQKLMQVVPQET
(3) STEAP1 (six transmembrane epithelial antigen of prostate,
Genbank accession no. NM.sub.--012449 Cancer Res. 61 (15),
5857-5860 (2001), Hubert, R. S., et al. (1999) Proc. Natl. Acad.
Sci. USA. 96 (25):14523-14528); WO2004065577 (Claim 6);
WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (Claim
2); WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830
(Example 5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page
618-619); WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173,
Example 2; FIG. 2A); NP.sub.--036581 six transmembrane epithelial
antigen of the prostate Cross-references: MIM:604415;
NP.sub.--036581.1; NM.sub.--012449.sub.--1 339 aa
TABLE-US-00004 (SEQ ID NO 3)
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
VSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIV
VLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486 J. Biol.
Chem. 276 (29):27371-27375 (2001)); WO2004045553 (Claim 14);
WO200292836 (Claim 6; FIG. 12); WO200283866 (Claim 15; Page
116-121); US2003124140 (Example 16); US2003091580 (Claim 6);
WO200206317 (Claim 6; Page 400-408); Cross-references: GI:34501467;
AAK74120.3; AF361486.sub.--1 6995 aa
TABLE-US-00005 (SEQ ID NO: 4)
PVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEAMQPSTHTAVT
NVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSL
TSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDIS
TEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHS
EMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALL
TLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVI
YKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREIS
TSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRIS
TPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSR
GPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKT
TDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYS
SYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIH
SATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTES
TEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGEVPWLS
HPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSE
PETSSPPNLSSTSAEILATTEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSM
GITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPT
GATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPS
GATSQGTFTLDSSSTASWPGTHSATTQRFPRSVVTTPMSRGPEDVSWPSPLSVEKNSPPS
SLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNI
TSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSS
IRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPE
VSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQ
VTLPLGTSMTFLSGTHSTMSQGLSESEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLP
LTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIP
ATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVEDTTV
FTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLFGVPTSATTEVSMTEI
MSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLA
TTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSPFVEKTSSSSSLLSLPVTTSPS
VSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTE
KIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFET
TGFEAEPFSHLTSGLRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDW
PPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETIS
TGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSS
STFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSS
PILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQT
STSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASMQ
MASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSR
SGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGA
DVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTI
PRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPT
LTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNS
PGEPATTVSLVTHSAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEV
PGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVV
TSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVAS
SRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNS
TSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIP
ILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTFLVTSSRAVTSTTIPTLTISS
DEPETTTSLVTHSEAKMISAIPTLGVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPET
IDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPS
TVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVTHP
AVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTD
TSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPDASKMLTSLVISSGTDSTTT
EPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVS
TTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTIPN
FSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPG
EPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPD
ATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALL
STHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVD
LSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLT
VSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPP
KTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLA
SESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMV
PFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPE
KDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHR
SSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRK
FNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLN
REQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSS
LSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVG
PLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYT
LDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITN
LKYEEDMHRPGSRKFNTTERVLQTLVGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDA
ICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPG
TSTVDLGSGTPSSLPSPTSATAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQ
SLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELS
QLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGP
LLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLL
RPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGF
THRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPG
SRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSP
GLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGT
PSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNSSV
GPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPY
SLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTIT
NLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVD
TICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTP
GTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQ
GLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELS
QLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGP
LLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLL
RPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGF
THRNFVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPG
SRKFNTTERVLQGLLRPLFKNTSIGPLYSSCRLTLLRPEKDKAATRVDAICTHHPDPQSP
GLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGI
PPSLPETTATGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSV
GPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPY
TLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTII
NLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVD
AVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTP
DTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQ
GLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELS
QLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASP
LLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTLL
RPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGF
THRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGS
RKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPG
LDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTI
NNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRV
DLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTT
PKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRP
LFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHG
VTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQD
KVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHW
LGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRN
IEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFL
RMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGLITCLIC
GVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,
mesothelin, Genbank accession no. NM.sub.--005823 Yamaguchi, N., et
al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci.
USA. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. USA. 93
(1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995));
WO2003101283 (Claim 14); (WO2002102235 (Claim 13; Page 287-288);
WO2002101075 (Claim 4; Page 308-309); WO200271928 (Page 320-321);
WO9410312 (Page 52-57); Cross-references: MIM:601051;
NP.sub.--005814.2; NM.sub.--005823.sub.--1 622 aa
TABLE-US-00006 (SEQ ID NO: 5)
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS
LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL
DLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA
DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTW
SVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT
ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELY
PQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVK
GRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKA
RLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQ
KLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGT
PCLLGPGPVLTVLALLLASTLA
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34
(sodium phosphate), member 2, type II sodium-dependent phosphate
transporter 3b, Genbank accession no. NM.sub.--006424, J. Biol.
Chem. 277(22):19665-19672 (2002), Genomics 62 (2):281-284 (1999),
Feild, J. A., et al. (1999) Biochem. Biophys. Res. Commun. 258
(3):578-582); WO2004022778 (Claim 2); EP1394274 (Example 11);
WO2002102235 (Claim 13; Page 326); EP875569 (Claim 1; Page 17-19);
WO200157188 (Claim 20; Page 329); WO2004032842 (Example IV);
WO200175177 (Claim 24; Page 139-140); Cross-references: MIM:
604217; NP.sub.--006415.1; NM.sub.--006424.sub.--1 690 aa
TABLE-US-00007 (SEQ ID NO: 6)
MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKI
ELLPSYSTATLIDEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQ
GIGRLILLLGFLYFFVCSLDILSSAFQLVGGKMAGQFFSNSSIMSNPL
LGLVIGVLVTVLVQSSSTSTSIVVSMVSSSLLTVRAAIPIIMGANIGT
SITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHY
LEIITQLIVESFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAM
NDEKAKNKSLVKIWCKTFTNKTQINVTVPSTANCTSPSLCWTDGIQNW
TMKNVTYKENIAKCQHIFVNFHLPDLAVGTILLILSLLVLCGCLIMIV
KILGSVLKGQVATVIKKTINTDFPFPFAWLTGYLAILVGAGMTFIVQS
SSVFTSALTPLIGIGVITIERAYPLTLGSNIGTTTTAILAALASPGNA
LRSSLQIALCHFFFNISGILLWYPIPFTRLPIRMAKGLGNISAKYRWF
AVFYLIIFFFLIPLTVFGLSLAGWRVLVGVGVPVVFIIILVLCLRLLQ
SRCPRVLPKKLQNWNFLPLWMRSLKPWDAVVSKFTGCFQMRCCYCCRV
CCRACCLLCGCPKCCRCSKCCEDLEEAQEGQDVPVKAPETFDNITISR
EAQGEVPASDSKTECTAL
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type
1 and type 1-like), transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 5B, Genbank accession no. AB040878, Nagase T.,
et al. (2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1);
WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133
(Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20); WO2003101400
(Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;
HGNC:10737; 1093 aa
TABLE-US-00008 (SEQ ID NO: 7)
MVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCALSKHPTV
AFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANV
SLLQATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTN
AFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHNSTAVISSQGELYAA
TVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAY
FFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNC
SRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSA
ISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSL
QDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLY
IGTESGTILKALSTASRSLHGCYLEELHVLPPGRREPLRSLRILHSAR
ALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLE
DSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLC
RARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGI
GFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGS
WSKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTP
WTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPA
DGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFR
VRKRTCTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPC
SASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSP
WSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPVIL
PASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSCQHCQRQS
QESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANF
YPLQQTNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,
RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
US2003129192 (Claim 2); US2004044180 (Claim 12); US2004044179
(Claim 11); US2003096961 (Claim 11); US2003232056 (Example 5);
WO2003105758 (Claim 12); US2003206918 (Example 5); EP1347046 (Claim
1); WO2003025148 (Claim 20); Cross-references: GI:37182378;
AAQ88991.1; AY358628.sub.--1 141 aa
TABLE-US-00009 (SEQ ID NO: 8)
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTV
NVQDMCQKEVMEQSAGIMYRKSCASSAACLIASAGYQSFCSPGKLNSV
CISCCNTPLCNGPRPKKRGSSASALRPGLRTTILFLKLALFSAHC
(9) ETBR (Endothelin type B receptor, Genbank accession no.
AY275463); Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177,
34-39, 1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178,
248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992;
Arai H., et al. J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A.,
Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178, 656-663,
1991; Elshourbagy N. A., et al. J. Biol. Chem. 268, 3873-3879,
1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1-S4,
1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg R. L.,
et al. Proc. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; Bourgeois
C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto
Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij J. B., et
al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al.
Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al.
Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4,
2407-2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351-354,
1996; Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra
R. M. W., et al. Nat. Genet. 12, 445-447, 1996; Svensson P. J., et
al. Hum. Genet. 103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7,
115-124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, 198-206;
WO2004045516 (Claim 1); WO2004048938 (Example 2); WO2004040000
(Claim 151); WO2003087768 (Claim 1); WO2003016475 (Claim 1);
WO2003016475 (Claim 1); WO200261087 (FIG. 1); WO2003016494 (FIG.
6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page
124-125); EP522868 (Claim 8; FIG. 2); WO200177172 (Claim 1; Page
297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat.
No. 5,773,223 (Claim 1a; Col 31-34); WO2004001004; 442 aa
TABLE-US-00010 (SEQ ID NO: 9)
MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTP
PTKILWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEI
KETFKYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLA
LGDLLHIVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSL
CALSIDRYRAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGF
DIITMDYKGSYLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAI
TAFFYTLMTCEMLRKKSGMQIALNDHLKQRREVAKTVFCLVLVFALCW
LPLHLSRILKLTLYNQNDPNRCELLSFLLVLDYIGINMASLNSCINPI
ALYLVSKRFKNCFKSCLCCWCQSFEEKQSLEEKQSCLKFKANDHGYDN FRSSNKYSSS
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank
accession no. NM.sub.--017763); WO2003104275 (Claim 1);
WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074
(Claim 14; Page 61); WO2003018621 (Claim 1); WO2003024392 (Claim 2;
FIG. 93); WO200166689 (Example 6); Cross-references: LocusID:
54894; NP.sub.--060233.2; NM.sub.--017763.sub.--1 783 aa
TABLE-US-00011 (SEQ ID NO: 10)
MSGGHQLQLAALWPWLLMATLQAGFGRTGLVLAAAVESERSAEQKAII
RVIPLKMDPTGKLNLTLEGVFAGVAEITPAEGKLMQSHPLYLCNASDD
DNLEPGFISIVKLESPRRAPRPCLSLASKARMAGERGASAVLFDITED
RAAAEQLQQPLGLTWPVVLIWGNDAEKLMEFVYKNQKAHVRIELKEPP
AWPDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAI
SQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVI
SCLHEFHRNCVDPWLHQHRTCPLCVFNITEGDSFSQSLGPSRSYQEPG
RRLHLIRQHPGHAHYHLPAAYLLGPSRSAVARPPRPGPFLPSQEPGMG
PRHHRFPRAAHPRAPGEQQRLAGAQHPYAQGWGMSHLQSTSQHPAACP
VPLRRARPPDSSGSGESYCTERSGYLADGPASDSSSGPCHGSSSDSVV
NCTDISLQGVHGSSSTFCSSLSSDFDPLVYCSPKGDPQRVDMQPSVTS
RPRSLDSVVPTGETQVSSHVHYHRHRHHHYKKRFQWHGRKPGPETGVP
QSRPPIPRTQPQPEPPSPDQQVTGSNSAAPSGRLSNPQCPRALPEPAP
GPVDASSICPSTSSLFNLQKSSLSARHPQRKRRGGPSEPTPGSRPQDA
TVHPACQIFPHYTPSVAYPWSPEAHPLICGPPGLDKRLLPETPGPCYS
NSQPVWLCLTPRQPLEPHPPGEGPSEWSSDTAEGRPCPYPHCQVLSAQ
PGSEEELEELCEQAV
(11) STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2,
STMP, prostate cancer associated gene 1, prostate cancer associated
protein 1, six transmembrane epithelial antigen of prostate 2, six
transmembrane prostate protein, Genbank accession no. AF455138,
Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397
(Claim 1; FIG. 1); WO200272596 (Claim 13; Page 54-55); WO200172962
(Claim 1; FIG. 4B); WO2003104270 (Claim 11); WO2003104270 (Claim
16); US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612
(Claim 12; FIG. 10); WO200226822 (Claim 23; FIG. 2); WO200216429
(Claim 12; FIG. 10); Cross-references: GI:22655488; AAN04080.1;
AF455138.sub.--1 490 aa
TABLE-US-00012 (SEQ ID NO: 11)
MESISMMGSPKSLSETVLPNGINGIKDARKVTVGVIGSGDFAKSLTIR
LIRCGYHVVIGSRNPKFASEFFPHVVDVTHHEDALTKTNIIFVAIHRE
HYTSLWDLRHLLVGKILIDVSNNMRINQYPESNAEYLASLFPDSLIVK
GFNVVSAWALQLGPKDASRQVYICSNNIQARQQVIELARQLNFIPIDL
GSLSSAREIENLPLRLFTLWRGPVVVAISLATFFFLYSFVRDVIHPYA
RNQQSDFYKIPIEIVNKTLPIVAITLLSLVYLAGLLAAAYQLYYGTKY
RRFPPWLETWLQCRKQLGLLSETFAMVHVAYSLCLPMRRSERYLFLNM
AYQQVHANIENSWNEEEVWRIEMYISFGIMSLGLLSLLAVTSIPSVSN
ALNWREFSFIQSTLGYVALLISTFHVLIYGWKRAFEEEYYRFYTPPNF
VLALVLPSIVILGKIILFLPCISQKLKRIKKGWEKSQFLEEGIGGTIP HVSPERVTVM
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor
potential cation channel, subfamily M, member 4, Genbank accession
no. NM.sub.--017636 Xu, X. Z., et al. Proc. Natl. Acad. Sci. USA.
98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol.
Chem. 278 (33):30813-30820 (2003)); US2003143557 (Claim 4);
WO200040614 (Claim 14; Page 100-103); WO200210382 (Claim 1; FIG.
9A); WO2003042661 (Claim 12); WO200230268 (Claim 27; Page 391);
US2003219806 (Claim 4); WO200162794 (Claim 14; FIG. 1A-D);
Cross-references: MIM:606936; NP.sub.--060106.2;
NM.sub.--017636.sub.--1 1214 aa
TABLE-US-00013 (SEQ ID NO: 12)
MVVPEKEQSWIPKIFKKKTCTTFIVDSTDPGGTLCQCGRPRTAHPAVA
MEDAFGAAVVTVWDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDR
TDPAAVYSLVTRTWGFRAPNLVVSVLGGSGGPVLQTWLQDLLRRGLVR
AAQSTGAWIVTGGLHTGIGRHVGVAVRDHQMASTGGTKVVAMGVAPWG
VVRNRDTLINPKGSFPARYRWRGDPEDGVQFPLDYNYSAFFLVDDGTH
GCLGGENRFRLRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMLTRI
ENATQAQLPCLLVAGSGGAADCLAETLEDTLAPGSGGARQGEARDRIR
RFFPKGDLEVLQAQVERIMTRKELLTVYSSEDGSEEFETIVLKALVKA
CGSSEASAYLDELRLAVAWNRVDIAQSELFRGDIQWRSFHLEASLMDA
LLNDRPEFVRLLISHGLSLGHFLTPMRLAQLYSAAPSNSLIRNLLDQA
SHSAGTKAPALKGGAAELRPPDVGHVLRMLLGKMCAPRYPSGGAWDPH
PGQGFGESMYLLSDKATSPLSLDAGLGQAPWSDLLLWALLLNRAQMAM
YFWEMGSNAVSSALGACLLLRVMARLEPDAEEAARRKDLAFKFEGMGV
DLFGECYRSSEVRAARLLLRRCPLWGDATCLQLAMQADARAFFAQDGV
QSLLTQKWWGDMASTTPIWALVLAFFCPPLIYTRLITFRKSEEEPTRE
ELEFDMDSVINGEGPVGTADPAEKTPLGVPRQSGRPGCCGGRCGGRRC
LRRWFHFWGAPVTIFMGNVVSYLLFLLLFSRVLLVDFQPAPPGSLELL
LYFWAFTLLCEELRQGLSGGGGSLASGGPGPGHASLSQRLRLYLADSW
NQCDLVALTCFLLGVGCRLTPGLYHLGRTVLCIDFMVFTVRLLHIFTV
NKQLGPKIVIVSKMMKDVFFFLFFLGVWLVAYGVATEGLLRPRDSDFP
SILRRVFYRPYLQIFGQIPQEDMDVALMEHSNCSSEPGFWAHPPGAQA
GTCVSQYANWLVVLLLVIFLLVANILLVNLLIAMFSYTFGKVQGNSDL
YWKAQRYRLIREFHSRPALAPPFIVISHLRLLLRQLCRRPRSPQPSSP
ALEHFRVYLSKEAERKLLTWESVHKENFLLARARDKRESDSERLKRTS
QKVDLALKQLGHIREYEQRLKVLEREVQQCSRVLGWVAEALSRSALLP PGGPPPPDLPGSKD
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived
growth factor, Genbank accession no. NP.sub.--003203 or
NM.sub.--003212, Ciccodicola, A., et al. EMBO J. 8 (7):1987-1991
(1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003224411
(Claim 1); WO2003083041 (Example 1); WO2003034984 (Claim 12);
WO200288170 (Claim 2; Page 52-53); WO2003024392 (Claim 2; FIG. 58);
WO200216413 (Claim 1; Page 94-95, 105); WO200222808 (Claim 2; FIG.
1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18); U.S. Pat. No.
5,792,616 (FIG. 2); Cross-references: MIM:187395;
NP.sub.--003203.1; NM.sub.--003212.sub.--1 188 aa
TABLE-US-00014 (SEQ ID NO: 13)
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRD
DSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCA
CPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQA
FLPGCDGLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr
virus receptor) or Hs.73792 Genbank accession no. M26004, Fujisaku
et al. (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al.
J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl.
Acad. Sci. USA. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol.
35, 1025-1031, 1998; Weis J. J., et al. Proc. Natl. Acad. Sci. USA.
83, 5639-5643, 1986; Sinha S. K., et al. (1993) J. Immunol. 150,
5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1);
WO2003062401 (Claim 9); WO2004045520 (Example 4); WO9102536 (FIG.
9.1-9.9); WO2004020595 (Claim 1); Accession: P20023; Q13866;
Q14212; EMBL; M26004; AAA35786.1. 1033 aa
TABLE-US-00015 (SEQ ID NO: 14)
MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIR
YSCSGTFRLIGEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIV
PGGYKIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQANNMWGPTRLP
TCVSVFPLECPALPMIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGE
KIINCLSSGKWSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANF
FCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPILNGRHI
GNSLANVSYGSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTWS
GPAPRCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFT
LKGSKQIRCNAQGTWEPSAPVCEKECQAPPNILNGQKEDRHMVRFDPG
TSIKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACEATGRQLLT
KPQHQFVRPDVNSSCGEGYKLSGSVYQECQGTIPWFMEIRLCKEITCP
PPPVIYNGAHTGSSLEDFPYGTTVTYTCNPGPERGVEFSLIGESTIRC
TSNDQERGTWSGPAPLCKLSLLAVQCSHVHIANGYKISGKEAPYFYND
TVTFKCYSGFTLKGSSQIRCKADNTWDPEIPVCEKETCQHVRQSLQEL
PAGSRVELVNTSCQDGYQLTGHAYQMCQDAENGIWFKKIPLCKVIHCH
PPPVIVNGKHTGMMAENFLYGNEVSYECDQGFYLLGEKKLQCRSDSKG
HGSWSGPSPQCLRSPPVTRCPNPEVKHGYKLNKTHSAYSHNDIVYVDC
NPGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIGCPPPPKTPNGNHTG
WGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTSQPAPHCKEVNC
SSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGSPQSQCQSDHQ
WNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRERNYYT
DTSQKEAFHLEAREVYSVDPYNPAS
(15) CD79b (CD79B, CD79.beta., IGb (immunoglobulin-associated
beta), B29, Genbank accession no. NM.sub.--000626 or 11038674,
Proc. Natl. Acad. Sci. USA. (2003) 100 (7):4126-4131, Blood (2002)
100 (9):3068-3076, Muller et al. (1992) Eur. J. Immunol. 22
(6):1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768,
US2004101874 (claim 1, page 102); WO2003062401 (claim 9);
WO200278524 (Example 2); US2002150573 (claim 5, page 15); U.S. Pat.
No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO
99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B);
WO200055351 (claim 11, pages 1145-1146); Cross-references:
MIM:147245; NP.sub.--000617.1; NM.sub.--000626.sub.--1 229 aa
TABLE-US-00016 (SEQ ID NO: 15)
MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQ
SPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRM
EESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMG
FSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEE
DHIYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase
anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no.
NM.sub.--030764, Genome Res. 13 (10):2265-2270 (2003),
Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002),
Proc. Natl. Acad. Sci. USA. 98 (17):9772-9777 (2001), Xu, M. J., et
al. (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775;
WO2004016225 (Claim 2); WO2003077836; WO200138490 (Claim 5; FIG.
18D-1-18D-2); WO2003097803 (Claim 12); WO2003089624 (Claim 25);
Cross-references: MIM:606509; NP.sub.--110391.2;
NM.sub.--030764.sub.--1 508 aa
TABLE-US-00017 (SEQ ID NO: 16)
MLLWSLLVIFDAVTEQADSLTLVAPSSVFEGDSIVLKCQGEQNWKIQK
MAYHKDNKELSVFKKFSDFLIQSAVLSDSGNYFCSTKGQLFLWDKTSN
IVKIKVQELFQRPVLTASSFQPIEGGPVSLKCETRLSPQRLDVQLQFC
FFRENQVLGSGWSSSPELQISAVWSEDTGSYWCKAETVTHRIRKQSLQ
SQIHVQRIPISNVSLEIRAPGGQVTEGQKLILLCSVAGGTGNVTFSWY
REATGTSMGKKTQRSLSAELEIPAVKESDAGKYYCRADNGHVPIQSKV
VNIPVRIPVSRPVLTLRSPGAQAAVGDLLELHCEALRGSPPILYQFYH
EDVTLGNSSAPSGGGASFNLSLTAEHSGNYSCEANNGLGAQCSEAVPV
SISGPDGYRRDLMTAGVLWGLFGVLGFTGVALLLYALFHKISGESSAT
NEPRGASRPNPQEFTYSSPTPDMEELQPVYVNVGSVDVDVVYSQVWSM
QQPESSANIRTLLENKDSQVIYSSVKKS
(17) HER2 (ErbB2, Genbank accession no. M11730, Coussens L., et al.
Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature
319, 230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. USA.
82, 6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol. 165,
869-880, 2004; Kuhns J. J., et al. J. Biol. Chem. 274, 36422-36427,
1999; Cho H.-S., et al. Nature 421, 756-760, 2003; Ehsani A., et
al. (1993) Genomics 15, 426-429; WO2004048938 (Example 2);
WO2004027049 (FIG. 1I); WO2004009622; WO2003081210; WO2003089904
(Claim 9); WO2003016475 (Claim 1); US2003118592; WO2003008537
(Claim 1); WO2003055439 (Claim 29; FIG. 1A-B); WO2003025228 (Claim
37; FIG. 5C); WO200222636 (Example 13; Page 95-107); WO200212341
(Claim 68; FIG. 7); WO200213847 (Page 71-74); WO200214503 (Page
114-117); WO200153463 (Claim 2; Page 41-46); WO200141787 (Page 15);
WO200044899 (Claim 52; FIG. 7); WO200020579 (Claim 3; FIG. 2); U.S.
Pat. No. 5,869,445 (Claim 3; Col 31-38); WO9630514 (Claim 2; Page
56-61); EP1439393 (Claim 7); WO2004043361 (Claim 7); WO2004022709;
WO200100244 (Example 3; FIG. 4); Accession: P04626; EMBL; M11767;
AAA35808.1. EMBL; M11761; AAA35808.1. 1255 aa
TABLE-US-00018 (SEQ ID NO: 17)
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRH
LYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPL
QRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRS
LTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRAC
HPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQC
AAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGR
YTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSK
PCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGD
PASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIR
GRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTV
PWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQC
VNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTC
FGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQ
PCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFG
ILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETEL
RKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEIL
DEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGR
LGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDF
GLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTV
WELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWM
IDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSL
LEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHASSSTRSGG
GDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTH
DPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSP
REGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ
GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEY LGLDVPV
(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al
Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res.
Commun. 150, 89-96, 1988; Strausberg R. L., et al. Proc. Natl.
Acad. Sci. USA. 99:16899-16903, 2002; WO2004063709; EP1439393
(Claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661
(Claim 12); WO200278524 (Example 2); WO200286443 (Claim 27; Page
427); WO200260317 (Claim 2); Accession: P40199; Q14920; EMBL;
M29541; AAA59915.1. EMBL; M18728; 344 aa
TABLE-US-00019 (SEQ ID NO: 18)
MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEG
KEVLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYS
GRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPEL
PKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWVNGQSLPVSPRLQ
LSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVP
TISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPN
ITVNNSGSYMCQAHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIG VLARVALI
(19) MDP (DPEP1, Genbank accession no. BC017023, Proc. Natl. Acad.
Sci. USA. 99 (26):16899-16903 (2002)); WO2003016475 (Claim 1);
WO200264798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8);
WO9946284 (FIG. 9); Cross-references: MIM:179780; AAH17023.1;
BC017023.sub.--1 411 aa
TABLE-US-00020 (SEQ ID NO: 19)
MWSGWWLWPLVAVCTADFFRDEAERIMRDSPVIDGHNDLPWQLLDMFN
NRLQDERANLTTLAGTHTNIPKLRAGFVGGQFWSVYTPCDTQNKDAVR
RTLEQMDVVHRMCRMYPETFLYVTSSAGIRQAFREGKVASLIGVEGGH
SIDSSLGVLRALYQLGMRYLTLTHSCNTPWADNWLVDTGDSEPQSQGL
SPFGQRVVKELNRLGVLIDLAHVSVATMKATLQLSRAPVIFSHSSAYS
VCASRRNVPDDVLRLVKQTDSLVMVNFYNNYISCTNKANLSQVADHLD
HIKEVAGARAVGFGGDFDGVPRVPEGLEDVSKYPDLIAELLRRNWTEA
EVKGALADNLLRVFEAVEQASNLTQAPEEEPIPLDQLGGSCRTHYGYS
SGASSLHRHWGLLLASLAPLVLCLSLL
(20) IL20R.alpha. (IL20Ra, ZCYTOR7, Genbank accession no.
AF184971); Clark H. F., et al. Genome Res. 13, 2265-2270, 2003;
Mungall A. J., et al. Nature 425, 805-811, 2003; Blumberg H., et
al. Cell 104, 9-19, 2001; Dumoutier L., et al. J. Immunol. 167,
3545-3549, 2001; Parrish-Novak J., et al. J. Biol. Chem. 277,
47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry
42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172,
2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);
WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63);
WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261
(Page 57-59); WO200146232 (Page 63-65); WO9837193 (Claim 1; Page
55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971;
AAF01320.1. 553 aa
TABLE-US-00021 (SEQ ID NO: 20)
MRAPGRPALRPLPLPPLLLLLLAAPWGRAVPCVSGGLPKPANITFLSI
NMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCD
LSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVAL
TTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNR
TWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTL
KDQSSEFKAKIIFWYVLPISITVFLFSVMGYSIYRYIHVGKEKHPANL
ILIYGNEFDKRFFVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDV
SSLNDPQPSGNLRPPQEEEEVKHLGYASHLMEIFCDSEENTEGTSFTQ
QESLSRTIPPDKTVIEYEYDVRTTDICAGPEEQELSLQEEVSTQGTLL
ESQAALAVLGPQTLQYSYTPQLQDLDPLAQEHTDSEEGPEEEPSTTLV
DWDPQTGRLCIPSLSSFDQDSEGCEPSEGDGLGEEGLLSRLYEEPAPD
RPPGENETYLMQFMEEWGLYVQMEN
(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053) Gary S.
C., et al. Gene 256, 139-147, 2000; Clark H. F., et al. Genome Res.
13, 2265-2270, 2003; Strausberg R. L., et al. Proc. Natl. Acad.
Sci. USA. 99, 16899-16903, 2002; US2003186372 (Claim 11);
US2003186373 (Claim 11); US2003119131 (Claim 1; FIG. 52);
US2003119122 (Claim 1; FIG. 52); US2003119126 (Claim 1);
US2003119121 (Claim 1; FIG. 52); US2003119129 (Claim 1);
US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);
US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim
1); 911 aa
TABLE-US-00022 (SEQ ID NO: 21)
MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLGGALTIPCHVH
YLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDV
SLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGVVFLYREGSARYAFSFSGAQEACA
RIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGVV
DPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGLDH
CSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRENVYCFRDSAQPS
AIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSST
PEDPAEAPRTLLEFETQSMVPPTGESEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALW
AWPSELSSPGPEASLPTEPAAQEKSLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPT
ETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRA
PEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVPASGDCVPSPCHNGGT
CLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYG
AHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLS
GENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVL
RYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKAL
LIPPSSPMPGP
(22) EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession no.
NM.sub.--004442) Chan, J. and Watt, V. M., Oncogene 6 (6),
1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev.
Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000));
WO2003042661 (Claim 12); WO200053216 (Claim 1; Page 41);
WO2004065576 (Claim 1); WO2004020583 (Claim 9); WO2003004529 (Page
128-132); WO200053216 (Claim 1; Page 42); Cross-references:
MIM:600997; NP.sub.--004433.2; NM.sub.--004442.sub.--1 987 aa
TABLE-US-00023 (SEQ ID NO: 22)
MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDENMNTIRTYQ
VCNVFESSQNNWLRTKFIRRRGAHRIHVEMKFSVRDCSSIPSVPGSCKETFNLYYYEADF
DSATKTFPNWMENPWVKVDTIAADESFSQVDLGGRVMKINTEVRSFGPVSRSGFYLAFQD
YGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLY
CNGDGEWLVPIGRCMCKAGFEAVENGTVCRGCPSGTFKANQGDEACTHCPINSRTTSEGA
TNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLMLEWTPPRDSGGREDLVYNI
ICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAVNGVTDQSPF
SPQFASVNITTNQAAPSAVSIMHQVSRTVDSITLSWSQPDQPNGVILDYELQYYEKELSE
YNATAIKSPTNTVTVQGLKAGAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIQEK
LPLIIGSSAAGLVFLIAVVVIAIVCNRRRGFERADSEYTDKLQHYTSGHMTPGMKIYIDP
FTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEVCSGHLKLPGKREIFVAIKTLKSGY
TEKQRRDFLSEASIMGQFDHPNVIHLEGVVTKSTPVMIITEFMENGSLDSFLRQNDGQFT
VIQLVGMLRGIAAGMKYLADMNYVHRDLAARNILVNSNLVCKVSDFGLSRFLEDDTSDPT
YTSALGGKIPIRWTAPEAIQYRKFTSASDVWSYGIVMWEVMSYGERPYWDMTNQDVINAI
EQDYRLPPPMDCPSALHQLMLDCWQKDRNHRPKFGQIVNTLDKMIRNPNSLKAMAPLSSG
INLPLLDRTIPDYTSFNTVDEWLEAIKMGQYKESFANAGFTSFDVVSQMMMEDILRVGVT
LAGHQKKILNSIQVMRAQMNQIQSVEV
(23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899
(Claim 2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3);
US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143
(FIG. 60); WO2002102235 (Claim 13; Page 299); US2003091580 (Example
2); WO200210187 (Claim 6; FIG. 10); WO200194641 (Claim 12; FIG.
7b); WO200202624 (Claim 13; FIG. 1A-1B); US2002034749 (Claim 54;
Page 45-46); WO200206317 (Example 2; Page 320-321, Claim 34; Page
321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; FIG.
1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2;
Page 205-207); WO2004053079 (Claim 12); WO2003004989 (Claim 1);
WO200271928 (Page 233-234, 452-453); WO 0116318; 282 aa
TABLE-US-00024 (SEQ ID NO: 23)
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEP
DIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV
QLTDAGTYKCYIITSKGKKNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVV
WASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKV
TESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK
(24) PSCA (Prostate stem cell antigen precursor, Genbank accession
no. AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci. USA. 95,
1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000;
Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709;
EP1394274 (Example 11); US2004018553 (Claim 17); WO2003008537
(Claim 1); WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10;
Page 288); WO200140309 (Example 1; FIG. 17); US2001055751 (Example
1; FIG. 1b); WO200032752 (Claim 18; FIG. 1); WO9851805 (Claim 17;
Page 97); WO9851824 (Claim 10; Page 94); WO9840403 (Claim 2; FIG.
1B); Accession: O43653; EMBL; AF043498; AAC39607.1. 123 aa
TABLE-US-00025 (SEQ ID NO: 24)
MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLT
VISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGP
GQL
(25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGIC
fusion-partner-like protein/pid=AAP 14954.1-Homo sapiens Species:
Homo sapiens (human) WO2003054152 (Claim 20); WO2003000842 (Claim
1); WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45);
Cross-references: GI:30102449; AAP14954.1; AY260763.sub.--1 236
aa
TABLE-US-00026 (SEQ ID NO: 25)
MPGAAAAAAAAAAAMLPAQEAAKLYHTNYVRNSRAIGVLWAIFTICFAIVNVVCFIQPYW
IGDGVDTPQAGYFGLFHYCIGNGFSRELTCRGSFTDFSTLPSGAFKAASFFIGLSMMLII
ACIICFTLFFFCNTATVYKICAWMQLTSAACLVLGCMIFPDGWDSDEVKRMCGEKTDKYT
LGACSVRWAYILAIIGILDALILSFLAFVLGNRQDSLMAEELKAENKVLLSQYSLE
(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3,
BR3, Genbank accession No. NP.sub.--443177.1); NP.sub.--443177 BAFF
receptor/pid=NP.sub.--443177.1-Homo sapiens Thompson, J. S., et al.
Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611;
WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35; FIG.
6B); WO2003035846 (Claim 70; Page 615-616); WO200294852 (Col
136-137); WO200238766 (Claim 3; Page 133); WO200224909 (Example 3;
FIG. 3); Cross-references: MIM:606269; NP.sub.--443177.1;
NM.sub.--052945.sub.--1 184 aa
TABLE-US-00027 (SEQ ID NO: 26)
MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQ
ESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGD
KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAG
PEQQ
(27) CD22 (B-cell receptor CD22-B isoform, Genbank accession No.
NP-001762.1); Stamenkovic, I. and Seed, B., Nature 345 (6270),
74-77 (1990); US2003157113; US2003118592; WO2003062401 (Claim 9);
WO2003072036 (Claim 1; FIG. 1); WO200278524 (Example 2);
Cross-references: MIM:107266; NP.sub.--001762.1;
NM.sub.--001771.sub.--1 847 aa
TABLE-US-00028 (SEQ ID NO: 27)
MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFH
NPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLR
MESKTEKWMERIHLNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEG
VPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKH
TPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT
KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPL
PTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPK
KVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNT
TIACARCNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQ
FFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM
SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPHHSQKLRLEPVKVQHSGAYWCQ
GTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQG
LQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAES
SEMQRPPRTCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENV
DYVILKH
(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a
B cell-specific protein that covalently interacts with Ig beta
(CD79B) and forms a complex on the surface with Ig M molecules,
transduces a signal involved in B-cell differentiation) PROTEIN
SEQUENCE Full mpggpgv . . . dvqlekp (1.226; 226 aa), pI: 4.84, MW:
25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession No.
NP.sub.--001774.1; WO2003088808, US20030228319; WO2003062401 (claim
9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, FIG.
16); WO9207574 (FIG. 1); U.S. Pat. No. 5,644,033; Ha et al. (1992)
J. Immunol. 148(5):1526-1531; Mueller et al. (1992) Eur. J.
Biochem. 22:1621-1625; Hashimoto et al. (1994) Immunogenetics
40(4):287-295; Preud'homme et al. (1992) Clin. Exp. Immunol.
90(1):141-146; Yu et al. (1992) J. Immunol. 148(2) 633-637;
Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464; 226 aa
TABLE-US-00029 (SEQ ID NO: 28)
MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSN
NANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCG
TYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGD
EYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled
receptor that is activated by the CXCL13 chemokine, functions in
lymphocyte migration and humoral defense, plays a role in HIV-2
infection and perhaps development of AIDS, lymphoma, myeloma, and
leukemia) PROTEIN SEQUENCE Full mnypltl . . . atslttf (1.372; 372
aa), pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank
accession No. NP.sub.--001707.1; WO2004040000; WO2004015426;
US2003105292 (Example 2); U.S. Pat. No. 6,555,339 (Example 2);
WO200261087 (FIG. 1); WO200157188 (Claim 20, page 269); WO200172830
(pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2,
pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat. No.
5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);
WO9217497 (claim 7, FIG. 5); Dobner et al. (1992) Eur. J. Immunol.
22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779; 372
aa
TABLE-US-00030 (SEQ ID NO: 29)
MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSL
IFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTF
LCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLL
ALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVG
VVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSL
PVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSL
SESENATSLTTF
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)
that binds peptides and presents them to CD4+ T lymphocytes)
PROTEIN SEQUENCE Full mgsgwvp . . . vllpqsc (1.273; 273 aa, pI:
6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession
No. NP.sub.--002111.1; Tonnelle et al. (1985) EMBO J.
4(11):2839-2847; Jonsson et al. (1989) Immunogenetics
29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441;
Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903;
Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al.
(1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) Tissue Antigens
59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No. 6,153,408
(Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat. No.
6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics
30(1):66-68; Larhammar et al. (1985) J. Biol. Chem.
260(26):14111-14119; 273 aa
TABLE-US-00031 (SEQ ID NO: 30)
MGSGWVPWVVALLVNLTRLDSSMTQGTDSPEDFVIQAKADCYFTNGTEKVQFVVRFIFNL
EEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQAVDGVCRHNYRLGAPFTVGRK
VQPEVTVYPERTPLLHQHNLLHCSVTGFYPGDIKIKWFLNGQEERAGVMSTGPIRNGDWT
FQTVVMLEMTPELGHVYTCLVDHSSLLSPVSVEWRAQSEYSWRKMLSGIAAELLGLIFLL
VGIVIQLRAQKGYVRTQMSGNEVSRAVLLPQSC
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an
ion channel gated by extracellular ATP, may be involved in synaptic
transmission and neurogenesis, deficiency may contribute to the
pathophysiology of idiopathic detrusor instability) PROTEIN
SEQUENCE Full mgqagek . . . lephrst (1.422; 422 aa), pI: 7.63, MW:
47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.
NP.sub.--002552.2; Le et al. (1997) FEBS Lett. 418(1-2):195-199;
WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000)
Genome Res. 10:165-173; WO200222660 (claim 20); WO2003093444 (claim
1); WO2003087768 (claim 1); WO2003029277 (page 82); 422 aa
TABLE-US-00032 (SEQ ID NO: 31)
MGQAGCKGLCLSLFDYKTEKYVIAKNKKVGLLYRLLQASILAYLVVWVFLIKKGYQDVDT
SLQSAVITKVKGVAFTNTSDLGQRIWDVADYVIPAQGENVFFVVTNLIVTPNQRQNVCAE
NEGIPDGACSKDSDCHAGEAVTAGNGVKTGRCLRRENLARGTCEIFAWCPLETSSRPEEP
FLKEAEDFTIFIKNHIRFPKFNFSKSNVMDVKDRSFLKSCHFGPKNHYCPIFRLGSVIRW
AGSDFQDIALEGGVIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSVSSGYNFRFARY
YRDAAGVEFRTLMKAYGIRFDVMVNGKGAFFCDLVLIYLIKKREFYRDKKYEEVRGLEDS
SQEAEDEASGLGLSEQLTSGPGLLGMPEQQELQEPPEAKRGSSSQKGNGSVCPQLLEPHR ST
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN
SEQUENCE Full maeaity . . . tafrfpd (1.359; 359 aa), pI: 8.66, MW:
40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No.
NP.sub.--001773.1; WO2004042346 (claim 65); WO2003026493 (pages
51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al.
(1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002)
Proc. Natl. Acad. Sci. USA 99:16899-16903; 359 aa
TABLE-US-00033 (SEQ ID NO: 32)
MAEAITYADLRFVKAPLKKSISSRLGQDPGADDDGEITYENVQVPAVLGVPSSLASSVLG
DKAAVKSEQPTASWRAVTSPAVGRILPCRTTCLRYLLLGLLLTCLLLGVTAICLGVRYLQ
VSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQA
AEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMH
QKSCFYISLTSKNWQESQKQCETLSSKLATFSEIYPQSHSYYFLNSLLPNGGSGNSYWTG
LSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICEMTAFRFPD
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein
of the leucine rich repeat (LRR) family, regulates B-cell
activation and apoptosis, loss of function is associated with
increased disease activity in patients with systemic lupus
erythematosis) PROTEIN SEQUENCE Full mafdvsc . . . rwkyqhi (1.661;
661 aa), pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12,
Genbank accession No. NP.sub.--005573.1; US2002193567; WO9707198
(claim 11, pages 39-42); Miura et al. (1996) Genomics
38(3):299-304; Miura et al. (1998) Blood 92:2815-2822;
WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages
24-26); 661 aa
TABLE-US-00034 (SEQ ID NO: 33)
MAFDVSCFFWVVLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDTLPNTTEFLEF
SFNFLPTIHNRTFSRLMNLTFLDLTRCQINWIHEDTFQSHHQLSTLVLTGNPLIFMAETS
LNGPKSLKHLFLIQTGISNLEFIPVHNLENLESLYLGSNHISSIKFPKDFPARNLKVLDF
QNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIELGAFDSTVFQSLNFGGTPNLSVIFN
GLQNSTTQSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEHRFSDISSTTFQCFTQL
QELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDQLCQISAANFPSLTHLYIRGNVKKLH
LGVGCLEKLGNLQTLDLSHNDIEASDCCSLQLKNLSHLQTLNLSHNEPLGLQSQAFKECP
QLELLDLAFTRLHINAPQSPFQNLHFLQVLNLTYCFLDTSNQHLLAGLPVLRHLNLKGNH
FQDGTITKTNLLQTVGSLEVLILSSCGLLSIDQQAFHSLGKMSHVDLSHNSLTCDSIDSL
SHLKGIYLNLAANSINIISPRLLPILSQQSTINLSHNPLDCTCSNIHFLIWYKENLHKLE
GSEETTCANPPSLRGVKLSDVKLSCGITAIGIFFLIVFLLLLAILLFFAVKYLLRWKYQH I
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the
immunoglobulin Fc domain that contains C2 type Ig-like and ITAM
domains, may have a role in B-lymphocyte differentiation) PROTEIN
SEQUENCE Full mlprlll . . . vdyedam (1.429; 429 aa), pI: 5.28, MW:
46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.
NP.sub.--443170.1; WO2003077836; WO200138490 (claim 6, FIG.
18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci. USA
98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1);
WO2003089624 (claim 7); 429 aa
TABLE-US-00035 (SEQ ID NO: 34)
MLPRLLLLICAPLCEPAELFLIASPSHPTEGSPVTLTCKMPFLQSSDAQFQFCFFRDTRA
LGPGWSSSPKLQIAAMWKEDTGSYWCEAQTMASKVLRSRRSQINVHRVPVADVSLETQPP
GGQVMEGDRLVLICSVAMGTGDITFLWYKGAVGLNLQSKTQRSLTAEYEIPSVRESDAEQ
YYCVAENGYGPSPSGLVSITVRIPVSRPILMLRAPRAQAAVEDVLELHCEALRGSPPILY
WFYHEDITLGSRSAPSGGGASFNLSLTEEHSGNYSCEANNGLGAQRSEAVTLNFTVPTGA
RSNHLTSGVIEGLLSTLGPATVALLECYGLKRKIGRRSARDPLRSLPSPLPQEFTYLNSP
TPGQLQPIYENVNVVSGDEVYSLAYYNQPEQESVAAETLGTHMEDKVSLDIYSRLRKANI
TDVDYEDAM
(35) IRTA2 (Immunoglobulin superfamily receptor translocation
associated 2, a putative immunoreceptor with possible roles in B
cell development and lymphomagenesis; deregulation of the gene by
translocation occurs in some B cell malignancies) PROTEIN SEQUENCE
Full mllwvil . . . assaphr (1.977; 977 aa), pI: 6.88 MW: 106468 TM:
1 [P] Gene Chromosome: 1q21, Genbank accession No.
NP.sub.--112571.1; WO2003024392 (claim 2, FIG. 97); Nakayama et al.
(2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836;
WO200138490 (claim 3, FIG. 18B-1-18B-2); 977 aa
TABLE-US-00036 (SEQ ID NO: 35)
MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRFYSPQKTKWYHR
YLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDFSSASLILQAPLSVFEGDSVV
LRCRAKAEVTLNNTIYKNDNVLAFLNKRTDFHIPHACLKDNGAYRCTGYKESCCPVSSNT
VKIQVQEPFTRPVLRASSFQPISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWS
LSPNFQITAMWSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTLSPEKALNFE
GTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNYYCTADNG
LGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLHCEAQRGSLPILYQFHHEDAA
LERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSITVPVSHPVLTLSSA
EALTFEGATVTLHCEVQRGSPQILYQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNYY
CTADNGFGPQRSEVVSLFVTVPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWF
YHEDVTLGSSSAPSGGEASENLSLTAEHSGNYSCEANNGLVAQHSDTISLSVIVPVSRPI
LTFRAPRAQAVVGDLLELHCEALRGSSPILYWFYHEDVTLGKISAPSGGGASFNLSLTTE
HSGIYSCEADNGPEAQRSEMVTLKVAVPVSRPVLTLRAPGTHAAVGDLLELHCEALRGSP
LILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGL
TANRSGPFATGVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSPPDSDSQEPTYH
NVPAWEELQPVYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKVA
STPVSGSLFLASSAPHR
See also: WO04/045516 (3 Jun. 2004); WO03/000113 (3 Jan. 2003);
WO02/016429 (28 Feb. 2002); WO02/16581 (28 Feb. 2002); WO03/024392
(27 Mar. 2003); WO04/016225 (26 Feb. 2004); WO01/40309 (7 Jun.
2001), and U.S. Provisional patent application Ser. No. 60/520,842
"COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF
HEMATOPOIETIC ORIGIN", filed 17 Nov. 2003; all of which are
incorporated herein by reference in their entirety.
In an embodiment, the Ligand-Linker-Drug Conjugate has Formula
IIIa, where the Ligand is an antibody Ab including one that binds
at least one of CD30, CD40, CD70, Lewis Y antigen, w=0, y=0, and D
has Formula Ib. Exemplary Conjugates of Formula IIIa include where
R.sup.17 is --(CH.sub.2).sub.5--. Also included are such Conjugates
of Formula IIIa in which D has the structure of Compound 2 in
Example 3 and esters thereof. Also included are such Conjugates of
Formula IIIa containing about 3 to about 8, in one aspect, about 3
to about 5 Drug moieties D, that is, Conjugates of Formula Ia
wherein p is a value in the range about 3-8, for example about 3-5.
Conjugates containing combinations of the structural features noted
in this paragraph are also contemplated as within the scope of the
compounds of the invention.
In another embodiment, the Ligand-Linker-Drug Conjugate has Formula
IIIa, where Ligand is an Antibody Ab that binds one of CD30, CD40,
CD70, Lewis Y antigen, w=1, y=0, and D has Formula Ib. Included are
such Conjugates of Formula IIIa in which R.sup.17 is
--(CH.sub.2).sub.5--. Also included are such Conjugates of Formula
IIIa in which W is -Val-Cit-, and/or where D has the structure of
Compound 2 in Example 3 and esters thereof. Also included are such
Conjugates of Formula IIIc containing about 3 to about 8,
preferably about 3 to about 5 Drug moieties D, that is, Conjugates
of Formula Ia wherein p is a value in the range of about 3-8,
preferably about 3-5. Conjugates containing combinations of the
structural features noted in this paragraph are also exemplary.
In an embodiment, the Ligand-Linker-Drug Conjugate has Formula
IIIa, where the Ligand is an Antibody Ab that binds one of CD30,
CD40, CD70, Lewis Y antigen, w=1, y=1, and D has Formula Ib.
Included are Conjugates of Formula IIIc in which R.sup.17 is
--(CH.sub.2).sub.5--. Also included are such Conjugates of Formula
IIIa where: W is -Val-Cit-; Y has Formula X; D has the structure of
Compound 2 in Example 3 and esters thereof; p is about 3 to about
8, preferably about 3 to about 5 Drug moieties D. Conjugates
containing combinations of the structural features noted in this
paragraph are also contemplated within the scope of the compounds
of the invention.
A further embodiment is an antibody drug conjugate (ADC), or a
pharmaceutically acceptable salt or solvate thereof, wherein Ab is
an antibody that binds one of the tumor-associated antigens
(1)-(35) noted above (the "TAA Compound").
Another embodiment is the TAA Compound or pharmaceutically
acceptable salt or solvate thereof that is in isolated and purified
form.
Another embodiment is a method for killing or inhibiting the
multiplication of a tumor cell or cancer cell comprising
administering to a patient, for example a human with a
hyperproliferative disorder, an amount of the TAA Compound or a
pharmaceutically acceptable salt or solvate thereof, said amount
being effective to kill or inhibit the multiplication of a tumor
cell or cancer cell.
Another embodiment is a method for treating cancer comprising
administering to a patient, for example a human with a
hyperproliferative disorder, an amount of the TAA Compound or a
pharmaceutically acceptable salt or solvate thereof, said amount
being effective to treat cancer, alone or together with an
effective amount of an additional anticancer agent.
Another embodiment is a method for treating an autoimmune disease,
comprising administering to a patient, for example a human with a
hyperproliferative disorder, an amount of the TAA Compound or a
pharmaceutically acceptable salt or solvate thereof, said amount
being effective to treat an autoimmune disease.
The antibodies suitable for use in the invention can be produced by
any method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression, and
are preferably produced by recombinant expression techniques.
4.5.1 Production of Recombinant Antibodies
Antibodies of the invention can be produced using any method known
in the art to be useful for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression.
Recombinant expression of antibodies, or fragment, derivative or
analog thereof, requires construction of a nucleic acid that
encodes the antibody. If the nucleotide sequence of the antibody is
known, a nucleic acid encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., 1994, BioTechniques 17:242), which involves the
synthesis of overlapping oligonucleotides containing portions of
the sequence encoding the antibody, annealing and ligation of those
oligonucleotides, and then amplification of the ligated
oligonucleotides, e.g., by PCR.
Alternatively, a nucleic acid molecule encoding an antibody can be
generated from a suitable source. If a clone containing the nucleic
acid encoding the particular antibody is not available, but the
sequence of the antibody is known, a nucleic acid encoding the
antibody can be obtained from a suitable source (e.g., an antibody
cDNA library, or cDNA library generated from any tissue or cells
expressing the immunoglobulin) by, e.g., PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence.
If an antibody that specifically recognizes a particular antigen is
not commercially available (or a source for a cDNA library for
cloning a nucleic acid encoding such an immunoglobulin), antibodies
specific for a particular antigen can be generated by any method
known in the art, for example, by immunizing a patient, or suitable
animal model such as a rabbit or mouse, to generate polyclonal
antibodies or, more preferably, by generating monoclonal
antibodies, e.g., as described by Kohler and Milstein (1975, Nature
256:495-497) or, as described by Kozbor et al. (1983, Immunology
Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a
clone encoding at least the Fab portion of the antibody can be
obtained by screening Fab expression libraries (e.g., as described
in Huse et al., 1989, Science 246:1275-1281) for clones of Fab
fragments that bind the specific antigen or by screening antibody
libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane
et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid sequence encoding at least the variable domain
of the antibody is obtained, it can be introduced into a vector
containing the nucleotide sequence encoding the constant regions of
the antibody (see, e.g., International Publication No. WO 86/05807;
WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the
complete light or heavy chain that allow for the expression of a
complete antibody molecule are available. Then, the nucleic acid
encoding the antibody can be used to introduce the nucleotide
substitutions or deletion necessary to substitute (or delete) the
one or more variable region cysteine residues participating in an
intrachain disulfide bond with an amino acid residue that does not
contain a sulfhydyl group. Such modifications can be carried out by
any method known in the art for the introduction of specific
mutations or deletions in a nucleotide sequence, for example, but
not limited to, chemical mutagenesis and in vitro site directed
mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).
In addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.
81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. A chimeric antibody is a molecule in which
different portions are derived from different animal species, such
as those having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region, e.g.,
humanized antibodies.
Alternatively, techniques described for the production of single
chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science
242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be
adapted to produce single chain antibodies. Single chain antibodies
are formed by linking the heavy and light chain fragments of the Fv
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional Fv fragments
in E. coli may also be used (Skerra et al., 1988, Science
242:1038-1041).
Antibody fragments that recognize specific epitopes can be
generated by known techniques. For example, such fragments include,
but are not limited to the F(ab').sub.2 fragments that can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments that can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments.
Once a nucleic acid sequence encoding an antibody has been
obtained, the vector for the production of the antibody can be
produced by recombinant DNA technology using techniques well known
in the art. Methods that are well known to those skilled in the art
can be used to construct expression vectors containing the antibody
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. See, for example, the techniques described
in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual,
2.sup.nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in
Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an
antibody or the nucleotide sequence of an antibody can be
transferred to a host cell by conventional techniques (e.g.,
electroporation, liposomal transfection, and calcium phosphate
precipitation), and the transfected cells are then cultured by
conventional techniques to produce the antibody. In specific
embodiments, the expression of the antibody is regulated by a
constitutive, an inducible or a tissue, specific promoter.
The host cells used to express the recombinant antibody can be
either bacterial cells such as Escherichia coli, or, preferably,
eukaryotic cells, especially for the expression of whole
recombinant immunoglobulin molecule. In particular, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al.,
1990, BioTechnology 8:2).
A variety of host-expression vector systems can be utilized to
express the immunoglobulin antibodies. Such host-expression systems
represent vehicles by which the coding sequences of the antibody
can be produced and subsequently purified, but also represent cells
that can, when transformed or transfected with the appropriate
nucleotide coding sequences, express an antibody immunoglobulin
molecule in situ. These include, but are not limited to,
microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing immunoglobulin coding
sequences; yeast (e.g., Saccharomyces Pichia) transformed with
recombinant yeast expression vectors containing immunoglobulin
coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the
immunoglobulin coding sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing immunoglobulin coding sequences; or mammalian cell
systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
antibody being expressed. For example, when a large quantity of
such a protein is to be produced, vectors that direct the
expression of high levels of fusion protein products that are
readily purified might be desirable. Such vectors include, but are
not limited, to the E. coli expression vector pUR278 (Ruther et
al., 1983, EMBO J. 2:1791), in which the antibody coding sequence
may be ligated individually into the vector in frame with the lac Z
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the
like. pGEX Vectors can also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption and binding to a matrix
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis
virus (AcNPV) or the analogous virus from Drosophila Melanogaster
is used as a vector to express foreign genes. The virus grows in
Spodoptera frugzperda cells. The antibody coding sequence can be
cloned individually into non-essential regions (for example the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems
can be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest can be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) results in a
recombinant virus that is viable and capable of expressing the
immunoglobulin molecule in infected hosts. (e.g., see Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals can also be required for efficient translation
of inserted antibody coding sequences. These signals include the
ATG initiation codon and adjacent sequences. Furthermore, the
initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression can be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:51-544).
In addition, a host cell strain can be chosen to modulate the
expression of the inserted sequences, or modifies and processes the
gene product in the specific fashion desired. Such modifications
(e.g., glycosylation) and processing (e.g., cleavage) of protein
products can be important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
foreign protein expressed. To this end, eukaryotic host cells that
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include, but are not limited
to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483,
Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins,
stable expression is preferred. For example, cell lines that stably
express an antibody can be engineered. Rather than using expression
vectors that contain viral origins of replication, host cells can
be transformed with DNA controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of the foreign DNA,
engineered cells can be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci that in turn can be cloned
and expanded into cell lines. This method can advantageously be
used to engineer cell lines which express the antibody. Such
engineered cell lines can be particularly useful in screening and
evaluation of tumor antigens that interact directly or indirectly
with the antibody.
A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
DHFR, which confers resistance to methotrexate (Wigler et al.,
1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIB TECH 11(5):155-215) and hygro, which confers resistance
to hygromycin (Santerre et al., 1984, Gene 30:147). Methods
commonly known in the art of recombinant DNA technology which can
be used are described in Ausubel et al. (eds., 1993, Current
Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.
(eds), 1994, Current Protocols in Human Genetics, John Wiley &
Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).
The expression levels of an antibody can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use
of vectors based on gene amplification for the expression of cloned
genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press,
New York, 1987)). When a marker in the vector system expressing an
antibody is amplifiable, an increase in the level of inhibitor
present in culture of host cell will increase the number of copies
of the marker gene. Since the amplified region is associated with
the nucleotide sequence of the antibody, production of the antibody
will also increase (Crouse et al., 1983, Mol. Cell. Biol.
3:257).
The host cell can be co-transfected with two expression vectors,
the first vector encoding a heavy chain derived polypeptide and the
second vector encoding a light chain derived polypeptide. The two
vectors can contain identical selectable markers that enable equal
expression of heavy and light chain polypeptides. Alternatively, a
single vector can be used to encode both heavy and light chain
polypeptides. In such situations, the light chain should be placed
before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad.
Sci. USA 77:2197). The coding sequences for the heavy and light
chains can comprise cDNA or genomic DNA.
Once the antibody has been recombinantly expressed, it can be
purified using any method known in the art for purification of an
antibody, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after
Protein A, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins.
In yet another exemplary embodiment, the antibody is a monoclonal
antibody.
In any case, the hybrid antibodies have a dual specificity,
preferably with one or more binding sites specific for the hapten
of choice or one or more binding sites specific for a target
antigen, for example, an antigen associated with a tumor, an
autoimmune disease, an infectious organism, or other disease
state.
4.5.2 Production of Antibodies
The production of antibodies will be illustrated with reference to
anti-CD30 antibodies but it will be apparent for those skilled in
the art that antibodies to other members of the TNF receptor family
can be produced and modified in a similar manner. The use of CD30
for the production of antibodies is exemplary only and not intended
to be limiting.
The CD30 antigen to be used for production of antibodies may be,
e.g., a soluble form of the extracellular domain of CD30 or a
portion thereof, containing the desired epitope. Alternatively,
cells expressing CD30 at their cell surface (e.g., L540 (Hodgkin's
lymphoma derived cell line with a T cell phenotype) and L428
(Hodgkin's lymphoma derived cell line with a B cell phenotype)) can
be used to generate antibodies. Other forms of CD30 useful for
generating antibodies will be apparent to those skilled in the
art.
In another exemplary embodiment, the ErbB2 antigen to be used for
production of antibodies may be, e.g., a soluble form of the
extracellular domain of ErbB2 or a portion thereof, containing the
desired epitope. Alternatively, cells expressing ErbB2 at their
cell surface (e.g., NIH-3T3 cells transformed to overexpress ErbB2;
or a carcinoma cell line such as SK-BR-3 cells, see Stancovski et
al. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991)) can be used to
generate antibodies. Other forms of ErbB2 useful for generating
antibodies will be apparent to those skilled in the art.
(i) Polyclonal Antibodies
Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the
relevant antigen and an adjuvant. It may be useful to conjugate the
relevant antigen to a protein that is immunogenic in the species to
be immunized, e.g., keyhole limpet hemocyanin, serum albumin,
bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates,
or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g of the
protein or conjugate (for rabbits or mice, respectively) with 3
volumes of Freund's complete adjuvant and injecting the solution
intradermally at multiple sites. One month later the animals are
boosted with 1/5 to 1/10 the original amount of peptide or
conjugate in Freund's complete adjuvant by subcutaneous injection
at multiple sites. Seven to 14 days later the animals are bled and
the serum is assayed for antibody titer. Animals are boosted until
the titer plateaus. Preferably, the animal is boosted with the
conjugate of the same antigen, but conjugated to a different
protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
(ii) Monoclonal Antibodies
Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
In the hybridoma method, a mouse or other appropriate host animal,
such as a hamster, is immunized as hereinabove described to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support
stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies
produced by hybridoma cells is determined by immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA). The binding affinity
of the monoclonal antibody can, for example, be determined by the
Scatchard analysis of Munson et al., Anal. Biochem., 107:220
(1980).
After hybridoma cells are identified that produce antibodies of the
desired specificity, affinity, and/or activity, the clones may be
subcloned by limiting dilution procedures and grown by standard
methods (Goding, Monoclonal Antibodies: Principles and Practice,
pp. 59-103 (Academic Press, 1986)). Suitable culture media for this
purpose include, for example, D-MEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably
separated from the culture medium, ascites fluid, or serum by
conventional antibody purification procedures such as, for example,
protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188
(1992).
In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by substituting the
coding sequence for human heavy chain and light chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; and Morrison, et al. (1984) Proc. Natl. Acad. Sci. USA
81:6851), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
Typically such non-immunoglobulin polypeptides are substituted for
the constant domains of an antibody, or they are substituted for
the variable domains of one antigen-combining site of an antibody
to create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
(iii) Humanized Antibodies
A humanized antibody may have one or more amino acid residues
introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science 239:1534-1536 (1988)), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567) wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
The choice of human variable domains, both light and heavy, to be
used in making the humanized antibodies is very important to reduce
antigenicity. According to the so-called "best-fit" method, the
sequence of the variable domain of a rodent antibody is screened
against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
In another embodiment, the antibodies may be humanized with
retention of high affinity for the antigen and other favorable
biological properties. Humanized antibodies may be prepared by a
process of analysis of the parental sequences and various
conceptual humanized products using three-dimensional models of the
parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in
the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
Various forms of the humanized antibody are contemplated. For
example, the humanized antibody may be an antibody fragment, such
as a Fab. Alternatively, the humanized antibody may be an intact
antibody, such as an intact IgG1 antibody.
The Examples describe production of an exemplary humanized
anti-ErbB2 antibody. The humanized antibody may, for example,
comprise nonhuman hypervariable region residues incorporated into a
human variable heavy domain and may further comprise a framework
region (FR) substitution at a position selected from the group
consisting of 69H, 71H and 73H utilizing the variable domain
numbering system set forth in Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). In one embodiment, the
humanized antibody comprises FR substitutions at two or all of
positions 69H, 71H and 73H. Another Example describes preparation
of purified trastuzumab antibody from the HERCEPTIN.RTM.
formulation.
(iv) Human Antibodies
As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and
5,545,807.
Alternatively, phage display technology (McCafferty et al., Nature
348:552-553 (1990)) can be used to produce human antibodies and
antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905. As discussed above, human antibodies may also be
generated by in vitro activated B cells (see U.S. Pat. Nos.
5,567,610 and 5,229,275). Human anti-CD30 antibodies are described
in U.S. patent application Ser. No. 10/338,366.
(v) Antibody Fragments
Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example. Such
linear antibody fragments may be monospecific or bispecific.
(vi) Bispecific Antibodies
Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
CD30 protein. Alternatively, an anti-CD30 arm may be combined with
an arm which binds to a Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so
as to focus cellular defense mechanisms to the CD30-expressing
cell. Bispecific antibodies may also be used to localize cytotoxic
agents to cells which express CD30.
Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO 10:3655-3659 (1991).
According to a different approach, antibody variable domains with
the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain, fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
In one embodiment of this approach, the bispecific antibodies are
composed of a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. It was found that this asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For further details of generating
bispecific antibodies see, for example, Suresh et al., Methods in
Enzymology, 121:210 (1986).
According to another approach described in U.S. Pat. No. 5,731,168,
the interface between a pair of antibody molecules can be
engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the CH3 domain of an antibody constant
domain. In this method, one or more small amino acid side chains
from the interface of the first antibody molecule are replaced with
larger side chains (e.g., tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s)
are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g.,
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
Techniques for generating bispecific antibodies from antibody
fragments have also been described in the literature. For example,
bispecific antibodies can be prepared using chemical linkage.
Brennan et al., Science, 229: 81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab'-SH
fragments from E. coli, which can be chemically coupled to form
bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225
(1992) describe the production of a fully humanized bispecific
antibody F(ab').sub.2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected to directed chemical coupling
in vitro to form the bispecific antibody.
Various techniques for making and isolating bispecific antibody
fragments directly from recombinant cell culture have also been
described. For example, bispecific antibodies have been produced
using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For
example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
(vii) Other Amino Acid Sequence Modifications
Amino acid sequence modification(s) of the antibodies described
herein are contemplated. For example, it may be desirable to
improve the binding affinity and/or other biological properties of
the antibody. Amino acid sequence variants of the antibodies are
prepared by introducing appropriate nucleotide changes into the
antibody nucleic acid, or by peptide synthesis. Such modifications
include, for example, deletions from, and/or insertions into and/or
substitutions of residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites.
A useful method for identification of certain residues or regions
of the antibody that are favored locations for mutagenesis is
called "alanine scanning mutagenesis" as described by Cunningham
and Wells Science, 244:1081-1085 (1989). Here, a residue or group
of target residues are identified (e.g., charged residues such as
arg, asp, his, lys, and glu) and replaced by a neutral or
negatively charged amino acid (most preferably alanine or
polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g., for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
Another type of variant is an amino acid substitution variant.
These variants have at least one amino acid residue in the antibody
molecule replaced by a different residue. The sites of greatest
interest for substitutional mutagenesis include the hypervariable
regions, but FR alterations are also contemplated.
Substantial modifications in the biological properties of the
antibody are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally-occurring residues are divided into
groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.
A particularly preferred type of substitutional variant involves
substituting one or more hypervariable region residues of a parent
antibody (e.g., a humanized or human antibody). Generally, the
resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and the antigen. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
It may be desirable to modify the antibody of the invention with
respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al. J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J.
Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug
Design 3:219-230 (1989).
To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
(viii) Glycosylation Variants
Antibodies in the ADC of the invention may be glycosylated at
conserved positions in their constant regions (Jefferis and Lund,
(1997) Chem. Immunol. 65:111-128; Wright and Morrison, (1997)
TibTECH 15:26-32). The oligosaccharide side chains of the
immunoglobulins affect the protein's function (Boyd et al., (1996)
Mol. Immunol. 32:1311-1318; Wittwe and Howard, (1990) Biochem.
29:4175-4180), and the intramolecular interaction between portions
of the glycoprotein which can affect the conformation and presented
three-dimensional surface of the glycoprotein (Hefferis and Lund,
supra; Wyss and Wagner, (1996) Current Opin. Biotech. 7:409-416).
Oligosaccharides may also serve to target a given glycoprotein to
certain molecules based upon specific recognition structures. For
example, it has been reported that in agalactosylated IgG, the
oligosaccharide moiety `flips` out of the inter-CH2 space and
terminal N-acetylglucosamine residues become available to bind
mannose binding protein (Malhotra et al., (1995) Nature Med.
1:237-243). Removal by glycopeptidase of the oligosaccharides from
CAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibody
which recognizes the CDw52 antigen of human lymphocytes) produced
in Chinese Hamster Ovary (CHO) cells resulted in a complete
reduction in complement mediated lysis (CMCL) (Boyd et al., (1996)
Mol. Immunol. 32:1311-1318), while selective removal of sialic acid
residues using neuraminidase resulted in no loss of DMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression of
.beta.(1,4)--N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al. (1999) Mature
Biotech. 17:176-180).
Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
Glycosylation variants of antibodies are variants in which the
glycosylation pattern of an antibody is altered. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, adding one or more carbohydrate moieties to the antibody,
changing the composition of glycosylation (glycosylation pattern),
the extent of glycosylation, etc.
Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it
contains one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites). The alteration may also be made
by the addition of, or substitution by, one or more serine or
threonine residues to the sequence of the original antibody (for
O-linked glycosylation sites). Similarly, removal of glycosylation
sites can be accomplished by amino acid alteration within the
native glycosylation sites of the antibody.
The amino acid sequence is usually altered by altering the
underlying nucleic acid sequence. These methods include, but are
not limited to, isolation from a natural source (in the case of
naturally-occurring amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the antibody.
The glycosylation (including glycosylation pattern) of antibodies
may also be altered without altering the amino acid sequence or the
underlying nucleotide sequence. Glycosylation largely depends on
the host cell used to express the antibody. Since the cell type
used for expression of recombinant glycoproteins, e.g., antibodies,
as potential therapeutics is rarely the native cell, significant
variations in the glycosylation pattern of the antibodies can be
expected. See, e.g., Hse et al., (1997) J. Biol. Chem.
272:9062-9070. In addition to the choice of host cells, factors
which affect glycosylation during recombinant production of
antibodies include growth mode, media formulation, culture density,
oxygenation, pH, purification schemes and the like. Various methods
have been proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261; 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example using endoglycosidase H (Endo H). In
addition, the recombinant host cell can be genetically engineered,
e.g., make defective in processing certain types of
polysaccharides. These and similar techniques are well known in the
art.
The glycosylation structure of antibodies can be readily analyzed
by conventional techniques of carbohydrate analysis, including
lectin chromatography, NMR, Mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides.
4.5.2a Screening for Antibody-Drug Conjugates (ADC)
Transgenic animals and cell lines are particularly useful in
screening antibody drug conjugates (ADC) that have potential as
prophylactic or therapeutic treatments of diseases or disorders
involving overexpression of proteins including Lewis Y, CD30, CD40,
and CD70. Transgenic animals and cell lines are particularly useful
in screening antibody drug conjugates (ADC) that have potential as
prophylactic or therapeutic treatments of diseases or disorders
involving overexpression of HER2 (U.S. Pat. No. 6,632,979).
Screening for a useful ADC may involve administering candidate ADC
over a range of doses to the transgenic animal, and assaying at
various time points for the effect(s) of the ADC on the disease or
disorder being evaluated. Alternatively, or additionally, the drug
can be administered prior to or simultaneously with exposure to an
inducer of the disease, if applicable. Candidate ADC may be
screened serially and individually, or in parallel under medium or
high-throughput screening format. The rate at which ADC may be
screened for utility for prophylactic or therapeutic treatments of
diseases or disorders is limited only by the rate of synthesis or
screening methodology, including detecting/measuring/analysis of
data.
One embodiment is a screening method comprising (a) transplanting
cells from a stable renal cell cancer cell line into a non-human
animal, (b) administering an ADC drug candidate to the non-human
animal and (c) determining the ability of the candidate to inhibit
the formation of tumors from the transplanted cell line.
Another embodiment is a screening method comprising (a) contacting
cells from a stable Hodgkin's disease cell line with an ADC drug
candidate and (b) evaluating the ability of the ADC candidate to
block ligand activation of CD40.
Another embodiment is a screening method comprising (a) contacting
cells from a stable Hodgkin's disease cell line with an ADC drug
candidate and (b) evaluating the ability of the ADC candidate to
induce cell death. In one embodiment the ability of the ADC
candidate to induce apoptosis is evaluated.
One embodiment is a screening method comprising (a) transplanting
cells from a stable cancer cell line into a non-human animal, (b)
administering an ADC drug candidate to the non-human animal and (c)
determining the ability of the candidate to inhibit the formation
of tumors from the transplanted cell line. The invention also
concerns a method of screening ADC candidates for the treatment of
a disease or disorder characterized by the overexpression of HER2
comprising (a) contacting cells from a stable breast cancer cell
line with a drug candidate and (b) evaluating the ability of the
ADC candidate to inhibit the growth of the stable cell line.
Another embodiment is a screening method comprising (a) contacting
cells from a stable cancer cell line with an ADC drug candidate and
(b) evaluating the ability of the ADC candidate to block ligand
activation of HER2. In one embodiment the ability of the ADC
candidate to block heregulin binding is evaluated. In another
embodiment the ability of the ADC candidate to block
ligand-stimulated tyrosine phosphorylation is evaluated.
Another embodiment is a screening method comprising (a) contacting
cells from a stable cancer cell line with an ADC drug candidate and
(b) evaluating the ability of the ADC candidate to induce cell
death. In one embodiment the ability of the ADC candidate to induce
apoptosis is evaluated.
Another embodiment is a screening method comprising (a)
administering an ADC drug candidate to a transgenic non-human
mammal that overexpresses in its mammary gland cells a native human
HER2 protein or a fragment thereof, wherein such transgenic mammal
has stably integrated into its genome a nucleic acid sequence
encoding a native human HER2 protein or a fragment thereof having
the biological activity of native human HER2, operably linked to
transcriptional regulatory sequences directing its expression to
the mammary gland, and develops a mammary tumor not responding or
poorly responding to anti-HER2 antibody treatment, or to a
non-human mammal bearing a tumor transplanted from said transgenic
non-human mammal; and (b) evaluating the effect of the ADC
candidate on the target disease or disorder. Without limitations,
the disease or disorder may be a HER2-overexpressing cancer, such
as breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic and bladder cancer. The cancer
preferably is breast cancer which expressed HER2 in at least about
500,000 copies per cell, more preferably at least about 2,000,000
copies per cell. ADC drug candidates may, for example, be evaluated
for their ability to induce cell death and/or apoptosis, using
assay methods well known in the art and described hereinafter.
In one embodiment, candidate ADC are screened by being administered
to the transgenic animal over a range of doses, and evaluating the
animal's physiological response to the compounds over time.
Administration may be oral, or by suitable injection, depending on
the chemical nature of the compound being evaluated. In some cases,
it may be appropriate to administer the compound in conjunction
with co-factors that would enhance the efficacy of the compound. If
cell lines derived from the subject transgenic animals are used to
screen for compounds useful in treating various disorders, the test
compounds are added to the cell culture medium at an appropriate
time, and the cellular response to the compound is evaluated over
time using the appropriate biochemical and/or histological assays.
In some cases, it may be appropriate to apply the compound of
interest to the culture medium in conjunction with co-factors that
would enhance the efficacy of the compound.
Thus, provided herein are assays for identifying ADC which
specifically target and bind a target protein, the presence of
which is correlated with abnormal cellular function, and in the
pathogenesis of cellular proliferation and/or differentiation that
is causally related to the development of tumors.
To identify an ADC which blocks ligand activation of an ErbB (e.g.,
ErbB2) receptor, the ability of the compound to block ErbB ligand
binding to cells expressing the ErbB (ErbB2) receptor (e.g., in
conjugation with another ErbB receptor with which the ErbB receptor
of interest forms an ErbB hetero-oligomer) may be determined. For
example, cells isolated from the transgenic animal overexpressing
HER2 and transfected to express another ErbB receptor (with which
HER2 forms hetero-oligomer) may be incubated, i.e. culturing, with
the ADC and then exposed to labeled ErbB ligand. The ability of the
compound to block ligand binding to the ErbB receptor in the ErbB
hetero-oligomer may then be evaluated.
For example, inhibition of heregulin (HRG) binding to breast tumor
cell lines, overexpressing HER2 and established from the transgenic
non-human mammals (e.g., mice) herein, by the candidate ADC may be
performed using monolayer cultures on ice in a 24-well-plate
format. Anti-ErbB2 monoclonal antibodies may be added to each well
and incubated for 30 minutes. .sup.125I-labeled
rHRG.beta.1.sub.177-224 (25,000 cpm) may then be added, and the
incubation may be continued for 4 to 16 hours. Dose response curves
may be prepared and an IC.sub.50 value (cytotoxic activity) may be
calculated for the compound of interest.
Alternatively, or additionally, the ability of an ADC to block ErbB
ligand-stimulated tyrosine phosphorylation of an ErbB receptor
present in an ErbB hetero-oligomer may be assessed. For example,
cell lines established from the transgenic animals herein may be
incubated with a test ADC and then assayed for ErbB
ligand-dependent tyrosine phosphorylation activity using an
anti-phosphotyrosine monoclonal antibody (which is optionally
conjugated with a detectable label). The kinase receptor activation
assay described in U.S. Pat. No. 5,766,863 is also available for
determining ErbB receptor activation and blocking of that activity
by the compound.
In one embodiment, one may screen for ADC which inhibit HRG
stimulation of p180 tyrosine phosphorylation in MCF7 cells
essentially as described below. For example, a cell line
established from a HER2-transgenic animal may be plated in 24-well
plates and the compound may be added to each well and incubated for
30 minutes at room temperature; then rHRG.beta..sub.1177-244 may be
added to each well to a final concentration of 0.2 nM, and the
incubation may be continued for about 8 minutes. Media may be
aspirated from each well, and reactions may be stopped by the
addition of 100 .mu.l of SDS sample buffer (5% SDS, 25 mM DTT, and
25 mM Tris-HCl, pH 6.8). Each sample (25 .mu.l) may be
electrophoresed on a 4-12% gradient gel (Novex) and then
electrophoretically transferred to polyvinylidene difluoride
membrane. Antiphosphotyrosine (at 1 .mu.g/ml) immunoblots may be
developed, and the intensity of the predominant reactive band at
M.sub.r-180,000 may be quantified by reflectance densitometry. An
alternate method to evaluate inhibition of receptor phosphorylation
is the KIRA (kinase receptor activation) assay of Sadick et al.
(1998) Jour. of Pharm. and Biomed. Anal. Some of the well
established monoclonal antibodies against HER2 that are known to
inhibit HRG stimulation of p180 tyrosine phosphorylation can be
used as positive control in this assay. A dose-response curve for
inhibition of HRG stimulation of p180 tyrosine phosphorylation as
determined by reflectance densitometry may be prepared and an
IC.sub.50 for the compound of interest may be calculated.
One may also assess the growth inhibitory effects of a test ADC on
cell lines derived from a HER2-transgenic animal, e.g., essentially
as described in Schaefer et al. (1997) Oncogene 15:1385-1394.
According to this assay, the cells may be treated with a test
compound at various concentrations for 4 days and stained with
crystal violet or the redox dye Alamar Blue. Incubation with the
compound may show a growth inhibitory effect on this cell line
similar to that displayed by monoclonal antibody 2C4 on MDA-MB-175
cells (Schaefer et al., supra). In a further embodiment, exogenous
HRG will not significantly reverse this inhibition.
To identify growth inhibitory compounds that specifically target an
antigen of interest, one may screen for compounds which inhibit the
growth of cancer cells overexpressing antigen of interest derived
from transgenic animals, the assay described in U.S. Pat. No.
5,677,171 can be performed. According to this assay, cancer cells
overexpressing the antigen of interest are grown in a 1:1 mixture
of F12 and DMEM medium supplemented with 10% fetal bovine serum,
glutamine and penicillin streptomycin. The cells are plated at
20,000 cells in a 35 mm cell culture dish (2 mls/35 mm dish) and
the test compound is added at various concentrations. After six
days, the number of cells, compared to untreated cells is counted
using an electronic COULTER.TM. cell counter. Those compounds which
inhibit cell growth by about 20-100% or about 50-100% may be
selected as growth inhibitory compounds.
To select for compounds which induce cell death, loss of membrane
integrity as indicated by, e.g., PI, trypan blue or 7AAD uptake may
be assessed relative to control. The PI uptake assay uses cells
isolated from the tumor tissue of interest of a transgenic animal.
According to this assay, the cells are cultured in Dulbecco's
Modified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with
10% heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the
assay is performed in the absence of complement and immune effector
cells. The cells are seeded at a density of 3.times.10.sup.6 per
dish in 100.times.20 mm dishes and allowed to attach overnight. The
medium is then removed and replaced with fresh medium alone or
medium containing various concentrations of the compound. The cells
are incubated for a 3-day time period. Following each treatment,
monolayers are washed with PBS and detached by trypsinization.
Cells are then centrifuged at 1200 rpm for 5 minutes at 4.degree.
C., the pellet resuspended in 3 ml cold Ca.sup.2+ binding buffer
(10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl.sub.2) and aliquoted
into 35 mm strainer-capped 12.times.75 mm tubes (1 ml per tube, 3
tubes per treatment group) for removal of cell clumps. Tubes then
receive PI (10 .mu.g/ml). Samples may be analyzed using a
FACSCAN.TM. flow cytometer and FACSCONVERT.TM. CellQuest software
(Becton Dickinson). Those compounds which induce statistically
significant levels of cell death as determined by PI uptake may be
selected as cell death-inducing compounds.
In order to select for compounds which induce apoptosis, an annexin
binding assay using cells established from the tumor tissue of
interest of the transgenic animal is performed. The cells are
cultured and seeded in dishes as discussed in the preceding
paragraph. The medium is then removed and replaced with fresh
medium alone or medium containing 10 .mu.g/ml of the antibody drug
conjugate (ADC). Following a three-day incubation period,
monolayers are washed with PBS and detached by trypsinization.
Cells are then centrifuged, resuspended in Ca.sup.2+ binding buffer
and aliquoted into tubes as discussed above for the cell death
assay. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1
.mu.g/ml). Samples may be analyzed using a FACSCAN.TM. flow
cytometer and FACSCONVERT.TM. CellQuest software (Becton
Dickinson). Those compounds which induce statistically significant
levels of annexin binding relative to control are selected as
apoptosis-inducing compounds.
4.5.3 In Vitro Cell Proliferation Assays
Generally, the cytotoxic or cytostatic activity of an antibody drug
conjugate (ADC) is measured by: exposing mammalian cells having
receptor proteins to the antibody of the ADC in a cell culture
medium; culturing the cells for a period from about 6 hours to
about 5 days; and measuring cell viability. Cell-based in vitro
assays were used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of
the ADC of the invention.
The in vitro potency of antibody drug conjugates was measured by a
cell proliferation assay (Example 18, FIGS. 7-10). The
CellTiter-Glo.RTM. Luminescent Cell Viability Assay is a
commercially available (Promega Corp., Madison, Wis.), homogeneous
assay method based on the recombinant expression of Coleoptera
luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and 5,700,670).
This cell proliferation assay determines the number of viable cells
in culture based on quantitation of the ATP present, an indicator
of metabolically active cells (Crouch et al. (1993) J. Immunol.
Meth. 160:81-88, U.S. Pat. No. 6,602,677). The CellTiter-Glo.RTM.
Assay was conducted in 96 well format, making it amenable to
automated high-throughput screening (HTS) (Cree et al. (1995)
AntiCancer Drugs 6:398-404). The homogeneous assay procedure
involves adding the single reagent (CellTiter-Glo.RTM. Reagent)
directly to cells cultured in serum-supplemented medium. Cell
washing, removal of medium and multiple pipetting steps are not
required. The system detects as few as 15 cells/well in a 384-well
format in 10 minutes after adding reagent and mixing. The cells may
be treated continuously with ADC, or they may be treated and
separated from ADC. Generally, cells treated briefly, i.e. 3 hours,
showed the same potency effects as continuously treated cells.
The homogeneous "add-mix-measure" format results in cell lysis and
generation of a luminescent signal proportional to the amount of
ATP present. The amount of ATP is directly proportional to the
number of cells present in culture. The CellTiter-Glo.RTM. Assay
generates a "glow-type" luminescent signal, produced by the
luciferase reaction, which has a half-life generally greater than
five hours, depending on cell type and medium used (FIG. 24).
Viable cells are reflected in relative luminescence units (RLU).
The substrate, Beetle Luciferin, is oxidatively decarboxylated by
recombinant firefly luciferase with concomitant conversion of ATP
to AMP and generation of photons. The extended half-life eliminates
the need to use reagent injectors and provides flexibility for
continuous or batch mode processing of multiple plates. This cell
proliferation assay can be used with various multiwell formats,
e.g., 96 or 384 well format. Data can be recorded by luminometer or
CCD camera imaging device. The luminescence output is presented as
relative light units (RLU), measured over time.
The anti-proliferative effects of antibody drug conjugates were
measured by the cell proliferation, in vitro cell killing assay
above against four different breast tumor cell lines (FIGS. 7-10).
IC.sub.50 values were established for SK-BR-3 and BT-474 which are
known to over express HER2 receptor protein. Table 2a shows the
potency (IC.sub.50) measurements of exemplary antibody drug
conjugates in the cell proliferation assay against SK-BR-3 cells.
Table 2b shows the potency (IC.sub.50) measurements of exemplary
antibody drug conjugates in the cell proliferation assay against
BT-474 cells.
Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab;
Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; Trastuzumab-MC-MMAF,
4.1 MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab;
Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; and
Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab did not inhibit the
proliferation of MCF-7 cells (FIG. 9).
Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab;
Trastuzumab-MC-vc-PAS-MMAE, 3.3 MMAE/Ab;
Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab;
Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab;
Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; and
Trastuzumab-MC-MMAF, 4.1 MMAF/Ab did not inhibit the proliferation
of MDA-MB-468 cells (FIG. 10).
MCF-7 and MDA-MB-468 cells do not overexpress HER2 receptor
protein. The anti-HER2 antibody drug conjugates of the invention
therefore show selectivity for inhibition of cells which express
HER2.
TABLE-US-00037 TABLE 2a SK-BR-3 cells Antibody Drug Conjugate H =
trastuzumab linked via a cysteine [cys] except where noted
IC.sub.50 (.mu.g ADC/ml) H-MC-MMAF, 4.1 MMAF/Ab 0.008 H-MC-MMAF,
4.8 MMAF/Ab 0.002 H-MC-vc-PAB-MMAE, 0.007 H-MC-vc-PAB-MMAE 0.015
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.0035-0.01 H-MC-vc-PAB-MMAF, 4.4
MMAF/Ab 0.006-0.007 H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab 0.006
H-MC-(N--Me)vc-PAB-MMAF, 3.9 MMAF/Ab 0.0035 H-MC-MMAF, 4.1 MMAF/Ab
0.0035 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.010 H-MC-vc-PAB-MMAF, 3.8
MMAF/Ab 0.007 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.015 H-MC-vc-PAB-MMAF,
3.7 MMAF/Ab. 0.010 H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab 0.0025 H-MC-MMAE,
8.8 MMAE/Ab 0.018 H-MC-MMAE, 4.6 MMAE/Ab 0.05
H-MC-(L)val-(L)cit-PAB-MMAE, 8.7 0.0003 MMAE/Ab
H-MC-(D)val-(D)cit-PAB-MMAE, 8.2 0.02 MMAE/Ab
H-MC-(D)val-(L)cit-PAB-MMAE, 8.4 0.0015 MMAE/Ab
H-MC-(D)val-(L)cit-PAB-MMAE, 3.2 0.003 MMAE/Ab H-Trastuzumab 0.083
H-vc-MMAE, linked via a lysine [lys] 0.002 H-phe-lys-MMAE, linked
via a lysine [lys] 0.0015 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.004
Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab 0.01 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab
0.01 4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab 0.02 Anti-TF
Fab-MC-vc-PAB-MMAE* --
TABLE-US-00038 TABLE 2b BT474 cells Antibody Drug Conjugate H =
trastuzumab linked via a cysteine [cys] IC.sub.50 (.mu.g ADC/ml)
H-MC-MMAF, 4.1 MMAF/Ab 0.008 H-MC-MMAF, 4.8 MMAF/Ab 0.002
H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.015 H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab
0.02-0.05 H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.01 H-MC-vc-PAB-MMAF, 4.8
MMAF/Ab 0.01 H-MC-vc-PAB-MMAE, 3.3 MMAE/Ab 0.02 H-MC-vc-PAB-MMAF,
3.7 MMAF/Ab. 0.02 H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.015
H-MC-(N--Me)vc-PAB-MMAF, 3.9 MMAF/Ab 0.010 H-MC-MMAF, 4.1 MMAF/Ab
0.00015 H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab 0.0025 H-MC-MMAE, 8.8 MMAE/Ab
0.04 H-MC-MMAE, 4.6 MMAE/Ab 0.07 4D5-Fc8-MC-vc-PAB-MMAF, 4.4
MMAF/Ab 0.008 Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab 0.01
7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab 0.015 4D5 Fab-MC-vc-PAB-MMAF, 1.5
MMAF/Ab 0.04 Anti-TF Fab-MC-vc-PAB-MMAE* -- H = trastuzumab 7C2 =
anti-HER2 murine antibody which binds a different epitope than
trastuzumab. Fc8 = mutant that does not bind to FcRn Hg =
"Hingeless" full-length humanized 4D5, with heavy chain hinge
cysteines mutated to serines. Expressed in E. coli (therefore
non-glycosylated.) Anti-TF Fab = anti-tissue factor antibody
fragment *activity against MDA-MB-468 cells
In a surprising and unexpected discovery, the in vitro cell
proliferation activity results of the ADC in Tables 2a and 2b show
generally that ADC with a low average number of drug moieties per
antibody showed efficacy, e.g., IC.sub.50<0.1 .mu.g ADC/ml. The
results suggest that at least for trastuzumab ADC, the optimal
ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5.
4.5.4 In Vivo Plasma Clearance and Stability
Pharmacokinetic plasma clearance and stability of ADC were
investigated in rats and cynomolgus monkeys. Plasma concentration
was measured over time. Table 2c shows pharmacokinetic data of
antibody drug conjugates and other dosed samples in rats. Rats are
a non-specific model for ErbB receptor antibodies, since the rat is
not known to express HER2 receptor proteins.
TABLE-US-00039 TABLE 2c Pharmacokinetics in Rats AUCinf CL T 1/2
Sample day* mL/day/ Cmax Term. % dose mg/kg .mu.g/mL kg .mu.g/mL
days Conj. H-MC-vc-PAB-MMAE 78.6 26.3 39.5 5.80 40.6 (Total Ab
H-MC-vc-PAB-MMAE 31.1 64.4 33.2 3.00 (Conj.) H-MC-vc-PAB-MMAF 170
12.0 47.9 8.4 50.0 (Total Ab) H-MC-vc-PAB-MMAF 83.9 24.0 44.7 4.01
(Conj.) H-MC-MMAE (Total Ab) 279 18.9 79.6 7.65 33 H-MC-MMAE
(Conj.) 90.6 62.9 62.9 4.46 5 mg/kg H-MC-MMAF (Total Ab) 299 6.74
49.1 11.6 37 H-MC-MMAF (Conj.) 110 18.26 50.2 4.54 H-MC-vc-MMAF,
306 6.6 78.7 11.9 19.6 wo/PAB, (Total Ab) H-MC-vc-MMAF, 59.9 33.4
82.8 2.1 wo/PAB, (Conj.) H-Me-vc-PAB-MMAF 186 10.8 46.9 8.3 45.3
(Total Ab) H-Me-vc-PAB-MMAF 84.0 23.8 49.6 4.3 (Conj.)
H-Me-vc-PAB-MMAE 135 15.0 44.9 11.2 23.8 (Total Ab)
H-Me-vc-PAB-MMAE 31.9 63.8 45.2 3.0 (Conj.) H-MC-vc-MMAF, 306 6.6
78.7 11.9 19.6 wo/PAB, (Total Ab) H-MC-vc-MMAF, 59.9 33.4 82.8 2.1
wo/PAB, (Conj.) H-MC-(D)val-(L)cit-PAB- 107 19.2 30.6 9.6 38.1 MMAE
(Total Ab) H-MC-(D)val-(L)cit-PAB- 40 50.4 33.7 3.98 MMAE (Conj.)
H-MC-(Me)-vc-PAB- 135.1 15.0 44.9 11.2 23.8 MMAE, Total Ab
H-MC-(Me)-vc-PAB- 31.9 63.8 45.2 2.96 MMAE, Conj.
H-MC-(D)val-(D)cit-PAB- 88.2 22.8 33.8 10.5 38.3 MMAE, Total Ab
H-MC-(D)val-(D)cit-PAB- 33.6 59.8 36.0 4.43 MMAE, Conj.
H-MC-vc-PAB-MMAE, 78.6 26.3 39.5 5.8 40.6 Total Ab
H-MC-vc-PAB-MMAE, 31.1 64.4 33.2 3.00 Conj. H linked to MC by
lysine [lys] MMAF 0.99 204 280 0.224 -- 200 .mu.g/kg MMAE 3.71 62.6
649 0.743 -- 206 .mu.g/kg HER F(ab').sub.2-MC-vc- 9.3 217 34.4 0.35
95 MMAE, Total Ab HER F(ab').sub.2-MC-vc- 8.8 227 36.9 0.29 MMAE,
Conj. 4D5-H-Fab-MC-vc- 43.8 46.2 38.5 1.49 68 MMAF, Total Ab
4D5-H-Fab-MC-vc- 29.9 68.1 34.1 1.12 MMAF, Conj. 4D5-H-Fab-MC-vc-
71.5 70.3 108 1.18 59 MMAE, Total Ab 4D5-H-Fab-MC-vc- 42.2 118.9
114 0.74 MMAE, Conj. 4D5-H-Fab 93.4 53.9 133 1.08 --
H-MC-vc-PAB-MMAF, 170 12.03 47.9 8.44 49.5 Total Ab
H-MC-vc-PAB-MMAF, 83.9 23.96 44.7 4.01 Conj. H-MC-vc-PAB-MMAF- 211
9.8 39.8 8.53 34.3 DMAEA, Total Ab H-MC-vc-PAB-MMAF- 71.5 28.2 38.8
3.64 DMAEA, Conj. H-MC-vc-PAB-MMAF- 209 9.75 53.2 8.32 29.7 TEG,
Total Ab H-MC-vc-PAB-MMAF- 63.4 31.8 34.9 4.36 TEG, Conj. H =
trastuzumab linked via a cysteine [cys] except where noted 2 mg/kg
dose except where noted
AUC inf is the area under the plasma concentration-time curve from
time of dosing to infinity and is a measure of the total exposure
to the measured entity (drug, ADC). CL is defined as the volume of
plasma cleared of the measured entity in unit time and is expressed
by normalizing to body weight. T1/2 term is the half-life of the
drug in the body measured during its elimination phase. The % Conj.
term is the relative amount of ADC compared to total antibody
detected, by separate ELISA immunoaffinity tests ("Analytical
Methods for Biotechnology Products", Ferraiolo et al, p85-98 in
Pharmacokinetics of Drugs (1994) P. G. Welling and L. P. Balant,
Eds., Handbook of Experimental Pharmacology, Vol. 110,
Springer-Verlag. The % Conj. calculation is simply AUCinf of
ADC/AUCinf total Ab, and is a general indicator of linker
stability, although other factors and mechanisms may be in
effect.
FIG. 11 shows a graph of a plasma concentration clearance study
after administration of the antibody drug conjugates:
H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to Sprague-Dawley rats.
Concentrations of total antibody and ADC were measured over
time.
FIG. 12 shows a graph of a two stage plasma concentration clearance
study where ADC was administered at different dosages and
concentrations of total antibody and ADC were measured over
time.
In Vivo Efficacy
The in vivo efficacy of the ADC of the invention was measured by a
high expressing HER2 transgenic explant mouse model. An allograft
was propagated from the Fo5 mmtv transgenic mouse which does not
respond to, or responds poorly to, HERCEPTIN.RTM. therapy. Subjects
were treated once with ADC and monitored over 3-6 weeks to measure
the time to tumor doubling, log cell kill, and tumor shrinkage.
Follow up dose-response and multi-dose experiments were
conducted.
Tumors arise readily in transgenic mice that express a mutationally
activated form of neu, the rat homolog of HER2, but the HER2 that
is overexpressed in breast cancers is not mutated and tumor
formation is much less robust in transgenic mice that overexpress
nonmutated HER2 (Webster et al. (1994) Semin. Cancer Biol.
5:69-76).
To improve tumor formation with nonmutated HER2, transgenic mice
were produced using a HER2 cDNA plasmid in which an upstream ATG
was deleted in order to prevent initiation of translation at such
upstream ATG codons, which would otherwise reduce the frequency of
translation initiation from the downstream authentic initiation
codon of HER2 (for example, see Child et al. (1999) J. Biol. Chem.
274: 24335-24341). Additionally, a chimeric intron was added to the
5' end, which should also enhance the level of expression as
reported earlier (Neuberger and Williams (1988) Nucleic Acids Res.
16: 6713; Buchman and Berg (1988) Mol. Cell. Biol. 8:4395; Brinster
et al. (1988) Proc. Natl. Acad. Sci. USA 85:836). The chimeric
intron was derived from a Promega vector, pCI-neo mammalian
expression vector (bp 890-1022). The cDNA 3'-end is flanked by
human growth hormone exons 4 and 5, and polyadenylation sequences.
Moreover, FVB mice were used because this strain is more
susceptible to tumor development. The promoter from MMTV-LTR was
used to ensure tissue-specific HER2 expression in the mammary
gland. Animals were fed the AIN 76A diet in order to increase
susceptibility to tumor formation (Rao et al. (1997) Breast Cancer
Res. and Treatment 45:149-158).
TABLE-US-00040 TABLE 2d Tumor measurements in allograft mouse model
- MMTV-HER2 Fo5 Mammary Tumor, athymic nude mice single dose at day
1 (T = 0) except where noted H = trastuzumab linked via a cysteine
[cys] except where noted Tumor Mean Sample doubling log Drugs time
cell per antibody Dose Ti PR CR (days) kill Vehicle 2-5 0
H-MC-vc-PAB-MMAE 1250 .mu.g/m.sup.2 5/5 4/7 0/7 18 1.5 8.7 MMAE/Ab
H-MC-vc-PAB-MMAF 555 .mu.g/m.sup.2 2/5 2/7 5/7 69 6.6 3.8 MMAF/Ab
H-MC(Me)-vc-PAB- >50 6.4 MMAF H-MC-MMAF 9.2 mg/kg 7/7 6/7 0/7 63
9 4.8 MMAF/Ab Ab 550 .mu.g/m.sup.2 at 0, 7, 14 and 21 days
H-MC-MMAF 14 mg/kg Ab 5/5 5/7 2/7 >63 4.8 MMAF/Ab 840
.mu.g/m.sup.2 at 0, 7, 14 and 21 days H-MC-vc-PAB-MMAF 3.5 mg/kg
5/6 1/7 3/7 >36 5.9 MMAF/Ab Ab 300 .mu.g/m.sup.2 at 0, 21, and
42 days H-MC-vc-PAB-MMAF 4.9 mg/kg 4/7 2/7 5/7 >90 5.9 MMAF/Ab
Ab 425 .mu.g/m.sup.2 at 0, 21, and 42 days H-MC-vc-PAB-MMAF 6.4
mg/kg 3/6 1/7 6/7 >90 5.9 MMAF/Ab Ab 550 .mu.g/m.sup.2 at 0, 21,
and 42 days H-(L)val-(L)cit-MMAE 10 mg/kg 7/7 1/7 0/7 15.2 1.1 8.7
MMAE/Ab H-MC-MMAE 10 mg/kg 7/7 0/7 0/7 4 0.1 4.6 MMAE/Ab
H-(D)val-(D)cit- 10 mg/kg 7/7 0/7 0/7 3 MMAE 4.2 MMAE/Ab
H-(D)val-(L)cit-MMAE 13 mg/kg 7/7 0/7 0/7 9 0.6 3.2 MMAE/Ab
H-MC(Me)-vc-MMAE 13 mg/kg 7/7 3/7 0/7 17 1.2 3.0 MMAE/Ab
H-(L)val-(D)cit-MMAE 12 mg/kg 7/7 0/7 0/7 5 0.2 3.5 MMAE/Ab
H-vc-MMAE 10 mg/kg 7/7 17 8.7 MMAE/Ab H-cys-vc-MMAF 1 mg/kg 7/7 3
3.8 MMAF/Ab H-cys-vc-MMAF 3 mg/kg 7/7 >17 3.8 MMAF/Ab
H-cys-vc-MMAF 10 mg/kg 4/7 4/7 3/7 >17 3.8 MMAF/Ab
H-MC-vc-MMAF-TEG 10 mg/kg 3/6 1/7 6/7 81 7.8 4 MMAF/Ab
H-MC-vc-MMAF-TEG 10 mg/kg 0/5 0/7 7/7 81 7.9 4 MMAF/Ab q3wk x 3
H-vc-MMAF (lot 1) 10 mg/kg 4/6 2/8 5/8 H-vc-MMAF (lot 2) 10 mg/kg
7/8 1/8 1/8 H-MC-MMAF 10 mg/kg 8/8 1/8 0/8 18 550 .mu.g/m.sup.2
H-(Me)-vc-MMAF 10 mg/kg 3/7 2/8 5/8 H-vc-MMAE 3.7 mg/kg at 6/6 0/7
1/7 17 2.3 7.5 MMAE/Ab 0, 7, 14, 21, 28 days H-vc-MMAE 7.5 mg/kg at
5/7 3/7 3/7 69 10 7.5 MMAE/Ab 0, 7, 14, 21, 28 days anti
IL8-vc-MMAE 7.5 mg/kg at 7/7 0/7 0/7 5 0.5 7.5 MMAE/Ab 0, 7, 14,
21, 28 days anti IL8-vc-MMAE 3.7 mg/kg at 6/6 0/7 0/7 3 0.2 7.5
MMAE/Ab 0, 7, 14, 21, 28 days H-fk-MMAE 7.5 mg/kg at 7/7 1/7 0/7 31
4.4 7.5 MMAE/Ab 0, 7, 14, 21, 28 days H-fk-MMAE 3.7 mg/kg at 7/7
0/7 0/7 8.3 0.9 7.5 MMAE/Ab 0, 7, 14, 21, 28 days anti IL8-fk-MMAE
7.5 mg/kg at 7/7 0/7 0/7 6 0.5 7.5 MMAE/Ab 0, 7, 14, 21, 28 days
anti IL8-fk-MMAE 3.7 mg/kg at 7/7 0/7 0/7 3 0.1 7.5 MMAE/Ab 0, 7,
14, 21, 28 days Trastuzumab 7.5 mg/kg at 7/7 0/7 0/7 5 0.4 0, 7,
14, 21, 28 days H-vc-MMAE 10 mg/kg 6/6 3/6 0/6 15 1.3 8.7 MMAE/Ab
1250 .mu.g/m.sup.2 H-vc-MMAE 10 mg/kg 7/7 5/7 >19 1250
.mu.g/m.sup.2 at 0, 7, and 14 days H-vc-MMAE 3 mg/kg at 7/7 8 0, 7,
and 14 days H-vc-MMAE 1 mg/kg at 7/7 7 0, 7, and 14 days H-vc-MMAF
10 mg/kg 8/8 5/8 >21 H-vc-MMAF 10 mg/kg at 4/7 4/7 3/7 >21 0,
7, and 14 days H-vc-MMAF 3 mg/kg at 7/7 6 0, 7, and 14 days
H-vc-MMAF 1 mg/kg at 8/8 4 0, 7, and 14 days Trastuzumab 10 mg/kg
at 8/8 3 0 and 7 days Hg-MC-vc-PAB- 10 mg/kg at 6/7 3/8 5/8 56 5.1
MMAF 0 days 4.1 MMAF/Ab Fc8-MC-vc-PAB- 10 mg/kg at 7/7 6/8 0/8 25
2.1 MMAF 0 days 4.4 MMAF/Ab 7C2-MC-vc-PAB- 10 mg/kg at 5/6 6/8 1/8
41 3.7 MMAF 0 days 4 MMAF/Ab H-MC-vc-PAB-MMAF 10 mg/kg at 3/8 3/8
5/8 62 5.7 5.9 MMAF/Ab 0 days 2H9-MC-vc-PAB- 9/9 >14 days MMAE
2H9-MC-vc-PAB- 9/9 >14 days MMAF 11D10-vc-PAB-MMAE 9/9 >14
days 11D10-vc-PAB-MMAF 9/9 11 days 7C2 = anti-HER2 murine antibody
which binds a different epitope than trastuzumab. Fc8 = mutant that
does not bind to FcRn Hg = "Hingeless" full-length humanized 4D5,
with heavy chain hinge cysteines mutated to serines. Expressed in
E. coli (therefore non-glycosylated.) 2H9 = Anti-EphB2R 11D10 =
Anti-0772P
The term Ti is the number of animals in the study group with tumor
at T=0/total animals in group. The term PR is the number of animals
attaining partial remission of tumor/ animals with tumor at T=0 in
group. The term CR is the number of animals attaining complete
remission of tumor/animals with tumor at T=0 in group. The term Log
cell kill is the time in days for the tumor volume to double-the
time in days for the control tumor volume to double divided by
3.32.times.time for tumor volume to double in control animals
(dosed with Vehicle). The log-cell-kill calculation takes into
account tumor growth delay resulting from treatment and tumor
volume doubling time of the control group. Anti-tumor activity of
ADC is classified with log-cell-kill values of:
++++.gtoreq.3.4 (highly active)
+++=2.5-3.4
++=1.7-2.4
+=1.0-1.6
inactive=0
FIG. 13 shows the mean tumor volume change over time in athymic
nude mice with MMTV-HER2Fo5 Mammary tumor allografts dosed on Day 0
with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 .mu.g/m.sup.2) and
Trastuzumab-MC-vc-PAB-MMAF (555 .mu.g/m.sup.2). (H=Trastuzumab).
The growth of tumors was retarded by treatment with ADC as compared
to control (Vehicle) level of growth. FIG. 14 shows the mean tumor
volume change over time in athymic nude mice with MMTV-HER2Fo5
Mammary tumor allografts dosed on Day 0 with 10 mg/kg (660
.mu.g/m.sup.2) of Trastuzumab-MC-MMAE and 1250 .mu.g/m.sup.2
Trastuzumab-MC-vc-PAB-MMAE. FIG. 15 shows the mean tumor volume
change over time in athymic nude mice with MMTV-HER2Fo5 Mammary
tumor allografts dosed with 650 .mu.g/m.sup.2 Trastuzumab-MC-MMAF.
Table 2d and FIGS. 13-15 show that the ADC have strong anti-tumor
activity in the allograft of a HER2 positive tumor (Fo5) that
originally arose in an MMTV-HER2 transgenic mouse. The antibody
alone (e.g., Trastuzumab) does not have significant anti-tumor
activity in this model (Erickson et al. U.S. Pat. No. 6,632,979).
As illustrated in FIGS. 13-15, the growth of the tumors was
retarded by treatment with ADC as compared to control (Vehicle)
level of growth.
In a surprising and unexpected discovery, the in vivo anti-tumor
activity results of the ADC in Table 2d show generally that ADC
with a low average number of drug moieties per antibody showed
efficacy, e.g., tumor doubling time>15 days and mean log cell
kill>1.0. FIG. 16 shows that for the antibody drug conjugate,
trastuzumab-MC-vc-PAB-MMAF, the mean tumor volume diminished and
did not progress where the MMAF:trastuzumab ratio was 2 and 4,
whereas tumor progressed at a ratio of 5.9 and 6, but at a rate
lower than Vehicle (buffer). The rate of tumor progression in this
mouse xenograft model was about the same, i.e. 3 days, for Vehicle
and trastuzumab. The results suggest that at least for trastuzumab
ADC, the optimal ratio of drug moieties per antibody may be less
than about 8, and may be about 2 to about 4.
4.5.5 Rodent Toxicity
Antibody drug conjugates and an ADC-minus control, "Vehicle", were
evaluated in an acute toxicity rat model. Toxicity of ADC was
investigated by treatment of male and female Sprague-Dawley rats
with the ADC and subsequent inspection and analysis of the effects
on various organs. Gross observations included changes in body
weights and signs of lesions and bleeding. Clinical pathology
parameters (serum chemistry and hematology), histopathology, and
necropsy were conducted on dosed animals.
It is considered that weight loss, or weight change relative to
animals dosed only with Vehicle, in animals after dosing with ADC
is a gross and general indicator of systemic or localized toxicity.
FIGS. 17-19 show the effects of various ADC and control (Vehicle)
after dosing on rat body weight.
Hepatotoxicity was measured by elevated liver enzymes, increased
numbers of mitotic and apoptotic figures and hepatocyte necrosis.
Hematolymphoid toxicity was observed by depletion of leukocytes,
primarily granuloctyes (neutrophils), and/or platelets, and
lymphoid organ involvement, i.e. atrophy or apoptotic activity.
Toxicity was also noted by gastrointestinal tract lesions such as
increased numbers of mitotic and apoptotic figures and degenerative
enterocolitis.
Enzymes indicative of liver injury that were studied include:
AST (aspartate aminotransferase)
Localization: cytoplasmic; liver, heart, skeletal muscle,
kidney
Liver:Plasma ratio of 7000:1
T1/2: 17 hrs
ALT (alanine aminotransferase)
Localization: cytoplasmic; liver, kidney, heart, skeletal
muscle
Liver:Plasma ratio of 3000:1 T1/2: 42 hrs; diurnal variation GGT
(g-glutamyl transferase) Localization: plasma membrane of cells
with high secretory or absorptive capacity; liver, kidney,
intestine Poor predictor of liver injury; commonly elevated in bile
duct disorders
The toxicity profiles of trastuzumab-MC-val-cit-MMAF,
trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and
trastuzumab-MC-val-cit-PAB-MMAF were studied in female
Sprague-Dawley rats (Example 19). The humanized trastuzumab
antibody does not bind appreciably to rat tissue, and any toxicity
would be considered non-specific. Variants at dose levels of 840
and 2105 ug/m.sup.2 MMAF were compared to
trastuzumab-MC-val-cit-PAB-MMAF at 2105 ug/m.sup.2.
Animals in groups 1, 2, 3, 4, 6, and 7 (Vehicle, 9.94 & 24.90
mg/kg trastuzumab-MC-val-cit-MMAF, 10.69 mg/kg
trastuzumab-MC(Me)-val-cit-PAB-MMAF, and 10.17 & 25.50 mg/kg
trastuzumab-MC-MMAF, respectively) gained weight during the study.
Animals in groups 5 and 8 (26.78 mg/kg
trastuzumab-MC(Me)-val-cit-PAB-MMAF and 21.85 mg/kg
trastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight during
the study. On Study Day 5, the change in body weights of animals in
groups 2, 6 and 7 were not significantly different from group 1
animals. The change in body weights of animals in groups 3, 4, 5
and 8 were statistically different from group 1 animals (Example
19).
Rats treated with trastuzumab-MC-MMAF (groups 6 and 7) were
indistinguishable from vehicle-treated control animals at both dose
levels; i.e. this conjugate showed a superior safety profile in
this model. Rats treated with trastuzumab-MC-val-cit-MMAF (without
the self-immolative PAB moiety; groups 2 and 3) showed
dose-dependent changes typical for MMAF conjugates; the extent of
the changes was less compared with a full length
MC-val-cit-PAB-MMAF conjugate (group 8). The platelet counts on day
5 were at approximately 30% of baseline values in animals of group
3 (high dose trastuzumab-MC-val-cit-MMAF) compared with 15% in
animals of group 8 (high dose trastuzumab-MC-val-cit-PAB-MMAF).
Elevation of liver enzymes AST and ALT, of bilirubin and the extent
of thrombocytopenia was most evident in animals treated with
trastuzumab-MC(Me)-val-cit-PAB-MMAF (groups 4 and 5) in a
dose-dependent fashion; animals of group 5 (high dose group) showed
on day 5 levels of ALT of approximately 10.times. the baseline
value and platelets were reduced by approximately 90% at the time
of necropsy.
Female Sprague Dawley Rats were also dosed at high levels (Example
19, High Dose study: Groups 2, 3, 4) with trastuzumab-MC-MMAF, and
Vehicle control (Group 1). Mild toxicity signals were observed,
including a dose-dependent elevation of liver enzymes ALT, AST and
GGT. On day 5 animals in the highest dose group showed a 2-fold
elevation of ALT and a 5-fold elevation of AST; GGT is also
elevated (6 U/L). Enzyme levels show a trend towards normalization
on day 12. There was a mild granulocytosis in all three dose groups
on day 5, the platelet count remained essentially unchanged in all
animals. Morphological changes were mild; animals treated at the
4210 .mu.g/m.sup.2 dose level (Group 2) showed unremarkable
histology of liver, spleen, thymus, intestines and bone marrow.
Mildly increased apoptotic and mitotic activity was observed in
thymus and liver, respectively in animals treated at the 5500
.mu.g/m.sup.2 dose level (Group 3). The bone marrow was
normocellular, but showed evidence of granulocytic hyperplasia,
which is consistent with the absolute granulocytosis observed in
the peripheral blood counts in these animals. Animals at the
highest dose in group 4 showed qualitatively the same features; the
mitotic activity in the liver appears somewhat increased compared
to animals in Group 3. Also, extramedullary hematopoiesis was seen
in spleen and liver.
EphB2R is a type 1 TM tyrosine kinase receptor with close homology
between mouse and human, and is over-expressed in colorectal cancer
cells. 2H9 is an antibody against EphB2R. The naked antibody has no
effect on tumor growth, but 2H9-val-cit-MMAE killed EphB2R
expressing cells and showed efficacy in a mouse xenograft model
using CXF1103 human colon tumors (Mao et al (2004) Cancer Res.
64:781-788). 2H9 and 7C2 are both mouse IgG1 anti-HER2 antibodies.
The toxicity profiles of 2H9-MC-val-cit-PAB-MMAF (3.7 MMAF/Ab),
7C2-MC-val-cit-PAB-MMAF (4 MMAF/Ab), and
trastuzumab-MC-val-cit-PAB-MMAF (5.9 MMAF/Ab) were compared. The
differences in the structure of each immunoconjugate or the drug
portion of the immunoconjugate may affect the pharmacokinetics and
ultimately the safety profile. The humanized trastuzumab antibody
does not bind appreciably to rat tissue, and any toxicity would be
considered non-specific.
Cynomolgus Monkey Toxicity/Safety
Similar to the rat toxicity/safety study, cynomolgus monkeys were
treated with ADC followed by liver enzyme measurements, and
inspection and analysis of the effects on various organs. Gross
observations included changes in body weights and signs of lesions
and bleeding. Clinical pathology parameters (serum chemistry and
hematology), histopathology, and necropsy were conducted on dosed
animals (Example 19).
The antibody drug conjugate, H-MC-vc-PAB-MMAE (H=trastuzumab linked
through cysteine) showed no evidence of liver toxicity at any of
the dose levels tested. Peripheral blood granulocytes showed
depletion after a single dose of 1100 mg/m.sup.2 with complete
recovery 14 days post-dose. The antibody drug conjugate
H-MC-vc-PAB-MMAF showed elevation of liver enzymes at 550
(transient) and 880 mg/m2 dose level, no evidence of
granulocytopenia, and a dose-dependent, transient (groups 2 &
3) decline of platelets.
4.6 Synthesis of the Compounds of the Invention
The Exemplary Compounds and Exemplary Conjugates can be made using
the synthetic procedures outlined below in FIGS. 25-36. As
described in more detail below, the Exemplary Compounds or
Exemplary Conjugates can be conveniently prepared using a Linker
having a reactive site for binding to the Drug and Ligand. In one
aspect, a Linker has a reactive site which has an electrophilic
group that is reactive to a nucleophilic group present on a Ligand,
such as but not limited to an antibody. Useful nucleophilic groups
on an antibody include but are not limited to, sulfhydryl, hydroxyl
and amino groups. The heteroatom of the nucleophilic group of an
antibody is reactive to an electrophilic group on a Linker and
forms a covalent bond to a Linker unit. Useful electrophilic groups
include, but are not limited to, maleimide and haloacetamide
groups. The electrophilic group provides a convenient site for
antibody attachment.
In another embodiment, a Linker has a reactive site which has a
nucleophilic group that is reactive to an electrophilic group
present on an antibody. Useful electrophilic groups on an antibody
include, but are not limited to, aldehyde and ketone carbonyl
groups. The heteroatom of a nucleophilic group of a Linker can
react with an electrophilic group on an antibody and form a
covalent bond to an antibody unit. Useful nucleophilic groups on a
Linker include, but are not limited to, hydrazide, oxime, amino,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide. The electrophilic group on an antibody provides a
convenient site for attachment to a Linker.
Carboxylic acid functional groups and chloroformate functional
groups are also useful reactive sites for a Linker because they can
react with secondary amino groups of a Drug to form an amide
linkage. Also useful as a reactive site is a carbonate functional
group on a Linker, such as but not limited to p-nitrophenyl
carbonate, which can react with an amino group of a Drug, such as
but not limited to N-methyl valine, to form a carbamate linkage.
Typically, peptide-based Drugs can be prepared by forming a peptide
bond between two or more amino acids and/or peptide fragments. Such
peptide bonds can be prepared, for example, according to the liquid
phase synthesis method (see E. Schroder and K. Lubke, "The
Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well
known in the field of peptide chemistry.
The synthesis of an illustrative Stretcher having an electrophilic
maleimide group is illustrated below in FIGS. 28 and 29. General
synthetic methods useful for the synthesis of a Linker are
described in FIG. 30. FIG. 31 shows the construction of a Linker
unit having a val-cit group, an electrophilic maleimide group and a
PAB self-immolative Spacer group. FIG. 32 depicts the synthesis of
a Linker having a phe-lys group, an electrophilic maleimide group,
with and without the PAB self-immolative Spacer group. FIG. 33
presents a general outline for the synthesis of a Drug-Linker
Compound, while FIG. 34 presents an alternate route for preparing a
Drug-Linker Compound. FIG. 35 depicts the synthesis of a branched
linker containing a BHMS group. FIG. 36 outlines the attachment of
an antibody to a Drug-Linker Compound to form a
Drug-Linker-Antibody Conjugate, and FIG. 34 illustrates the
synthesis of Drug-Linker-Antibody Conjugates having, for example
but not limited to, 2 or 4 drugs per Antibody.
As described in more detail below, the Exemplary Conjugates are
conveniently prepared using a Linker having two or more Reactive
Sites for binding to the Drug and a Ligand. In one aspect, a Linker
has a Reactive site which has an electrophilic group that is
reactive to a nucleophilic group present on a Ligand, such as an
antibody. Useful nucleophilic groups on an antibody include but are
not limited to, sulfhydryl, hydroxyl and amino groups. The
heteroatom of the nucleophilic group of an antibody is reactive to
an electrophilic group on a Linker and forms a covalent bond to a
Linker unit. Useful electrophilic groups include, but are not
limited to, maleimide and haloacetamide groups. The electrophilic
group provides a convenient site for antibody attachment.
In another embodiment, a Linker has a Reactive site which has a
nucleophilic group that is reactive to an electrophilic group
present on a Ligand, such as an antibody. Useful electrophilic
groups on an antibody include, but are not limited to, aldehyde and
ketone carbonyl groups. The heteroatom of a nucleophilic group of a
Linker can react with an electrophilic group on an antibody and
form a covalent bond to an antibody unit. Useful nucleophilic
groups on a Linker include, but are not limited to, hydrazide,
oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate,
and arylhydrazide. The electrophilic group on an antibody provides
a convenient site for attachment to a Linker.
4.6.1 Drug Moiety Synthesis
Typically, peptide-based Drugs can be prepared by forming a peptide
bond between two or more amino acids and/or peptide fragments. Such
peptide bonds can be prepared, for example, according to the liquid
phase synthesis method (see E. Schroder and K. Lubke, "The
Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well
known in the field of peptide chemistry.
The auristatin/dolastatin drug moieties may be prepared according
to the general methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No.
5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465;
Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; and Pettit
et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863.
In one embodiment, a Drug is prepared by combining about a
stoichiometric equivalent of a dipeptide and a tripeptide,
preferably in a one-pot reaction under suitable condensation
conditions. This approach is illustrated in FIGS. 25-27, below.
FIG. 25 illustrates the synthesis of an N-terminal tripeptide unit
F which is a useful intermediate for the synthesis of the drug
compounds of Formula Ib.
As illustrated in FIG. 25, a protected amino acid A (where PG
represents an amine protecting group, R.sup.4 is selected from
hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, alkyl-aryl,
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle,
alkyl-(C.sub.3-C.sub.8 heterocycle) wherein R.sup.5 is selected
from H and methyl; or R.sup.4 and R.sup.5 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from hydrogen, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and 6,
and form a ring with the carbon atom to which they are attached) is
coupled to t-butyl ester B (where R.sup.6 is selected from --H and
--C.sub.1-C.sub.8 alkyl; and R.sup.7 is selected from hydrogen,
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 carbocycle,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, alkyl-aryl,
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
alkyl-(C.sub.3-C.sub.8 heterocycle)) under suitable coupling
conditions, e.g., in the presence of PyBrop and
diisopropylethylamine, or using DCC (see, for example, Miyazaki, K.
et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).
Suitable protecting groups PG, and suitable synthetic methods to
protect an amino group with a protecting group are well known in
the art. See, e.g., Greene, T. W. and Wuts, P. G. M., Protective
Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley &
Sons. Exemplary protected amino acids A are PG-Ile and,
particularly, PG-Val, while other suitable protected amino acids
include, without limitation: PG-cyclohexylglycine,
PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid,
PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and
PG-tert-butylglycine. Z is an exemplary protecting group. Fmoc is
another exemplary protecting group. An exemplary t-butyl ester B is
dolaisoleuine t-butyl ester.
The dipeptide C can be purified, e.g., using chromatography, and
subsequently deprotected, e.g., using H.sub.2 and 10% Pd--C in
ethanol when PG is benzyloxycarbonyl, or using diethylamine for
removal of an Fmoc protecting group. The resulting amine D readily
forms a peptide bond with an amino acid BB (wherein R.sup.1 is
selected from --H, --C.sub.1-C.sub.8 alkyl and --C.sub.3-C.sub.8
carbocycle; and R.sup.2 is selected from --H and --C.sub.1-C.sub.8
alkyl; or R.sup.1 and R.sup.2 join, have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from --H, --C.sub.1-C.sub.8 alkyl and
--C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and
6, and form a ring with the nitrogen atom to which they are
attached; and R.sup.3 is selected from hydrogen, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle, --O--(C.sub.1-C.sub.8 alkyl),
-aryl, alkyl-aryl, alkyl-(C.sub.3-C.sub.8 carbocycle),
C.sub.3-C.sub.8 heterocycle and alkyl-(C.sub.3-C.sub.8
heterocycle)). N,N-Dialkyl amino acids are exemplary amino acids
for BB, such as commercially available N,N-dimethyl valine. Other
N,N-dialkyl amino acids can be prepared by reductive bis-alkylation
using known procedures (see, e.g., Bowman, R. E, Stroud, H. H J.
Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine
are two exemplary amino acids BB useful for the synthesis of
N-monoalkyl derivatives. The amine D and the amino acid BB react to
provide the tripeptide E using coupling reagent DEPC with
triethylamine as the base. The C-terminus protecting group of E is
subsequently deprotected using HCl to provide the tripeptide
compound of formula F.
Illustrative DEPC coupling methodology and the PyBrop coupling
methodology shown in FIG. 25 are outlined below in General
Procedure A and General Procedure B, respectively. Illustrative
methodology for the deprotection of a Z-protected amine via
catalytic hydrogenation is outlined below in General Procedure
C.
General Procedure A: Peptide Synthesis Using DEPC.
The N-protected or N,N-disubstituted amino acid or peptide D (1.0
eq.) and an amine BB (1.1 eq.) are diluted with an aprotic organic
solvent, such as dichloromethane (0.1 to 0.5 M). An organic base
such as triethylamine or diisopropylethylamine (1.5 eq.) is then
added, followed by DEPC (1.1 eq.). The resulting solution is
stirred, preferably under argon, for up to 12 hours while being
monitored by HPLC or TLC. The solvent is removed in vacuo at room
temperature, and the crude product is purified using, for example,
HPLC or flash column chromatography (silica gel column). Relevant
fractions are combined and concentrated in vacuo to afford
tripeptide E which is dried under vacuum overnight.
General Procedure B: Peptide Synthesis Using PyBrop.
The amino acid B (1.0 eq.), optionally having a carboxyl protecting
group, is diluted with an aprotic organic solvent such as
dichloromethane or DME to provide a solution of a concentration
between 0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is
added. Fmoc-, or Z-protected amino acid A (1.1 eq.) is added as a
solid in one portion, then PyBrop (1.2 eq.) is added to the
resulting mixture. The reaction is monitored by TLC or HPLC,
followed by a workup procedure similar to that described in General
Procedure A.
General Procedure C: Z-Removal Via Catalytic Hydrogenation.
Z-protected amino acid or peptide C is diluted with ethanol to
provide a solution of a concentration between 0.5 and 1.0 mM in a
suitable vessel, such as a thick-walled round bottom flask. 10%
palladium on carbon is added (5-10% w/w) and the reaction mixture
is placed under a hydrogen atmosphere. Reaction progress is
monitored using HPLC and is generally complete within 1-2 h. The
reaction mixture is filtered through a pre-washed pad of celite and
the celite is again washed with a polar organic solvent, such as
methanol after filtration. The eluent solution is concentrated in
vacuo to afford a residue which is diluted with an organic solvent,
preferably toluene. The organic solvent is then removed in vacuo to
afford the deprotected amine C.
FIG. 26 shows a method useful for making a C-terminal dipeptide of
formula K and a method for coupling the dipeptide of formula K with
the tripeptide of formula F to make drug compounds of Formula
Ib.
The dipeptide K can be readily prepared by condensation of the
modified amino acid Boc-Dolaproine G (see, for example, Pettit, G.
R., et al. Synthesis, 1996, 719-725), with an amine of formula H
using condensing agents well known for peptide chemistry, such as,
for example, DEPC in the presence of triethylamine, as shown in
FIG. 25.
The dipeptide of formula K can then be coupled with a tripeptide of
formula F using General Procedure D to make the Fmoc-protected drug
compounds of formula L which can be subsequently deprotected using
General Procedure E in order to provide the drug compounds of
formula (Ib).
General Procedure D: Drug Synthesis.
A mixture of dipeptide K (1.0 eq.) and tripeptide F (1 eq.) is
diluted with an aprotic organic solvent, such as dichloromethane,
to form a 0.1M solution, then a strong acid, such as
trifluoroacetic acid (1/2 v/v) is added and the resulting mixture
is stirred under a nitrogen atmosphere for two hours at 0.degree.
C. The reaction can be monitored using TLC or, preferably, HPLC.
The solvent is removed in vacuo and the resulting residue is
azeotropically dried twice, preferably using toluene. The resulting
residue is dried under high vacuum for 12 h and then diluted with
and aprotic organic solvent, such as dichloromethane. An organic
base such as triethylamine or diisopropylethylamine (1.5 eq.) is
then added, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.)
depending on the chemical functionality on the residue. The
reaction mixture is monitored by either TLC or HPLC and upon
completion, the reaction is subjected to a workup procedure similar
or identical to that described in General Procedure A.
General Procedure E: Fmoc-Removal Using Diethylamine.
An Fmoc-protected Drug L is diluted with an aprotic organic solvent
such as dichloromethane and to the resulting solution is added
diethylamine (1/2 v/v). Reaction progress is monitored by TLC or
HPLC and is typically complete within 2 h. The reaction mixture is
concentrated in vacuo and the resulting residue is azeotropically
dried, preferably using toluene, then dried under high vacuum to
afford Drug Ib having a deprotected amino group.
FIG. 27 shows a method useful for making MMAF derivatives of
Formula (Ib).
The dipeptide O can be readily prepared by condensation of the
modified amino acid Boc-Dolaproine G (see, for example, Pettit, G.
R., et al. Synthesis, 1996, 719-725), with a protected amino acid
of formula M using condensing agents well known for peptide
chemistry, such as, for example, DEPC in the presence of
triethylamine, as shown in FIGS. 25 and 26.
The dipeptide of formula O can then be coupled with a tripeptide of
formula F using General Procedure D to make the Fmoc-protected MMAF
compounds of formula P which can be subsequently deprotected using
General Procedure E in order to provide the MMAF drug compounds of
formula (Ib).
Thus, the above methods are useful for making Drugs that can be
used in the present invention.
4.6.2 Drug Linker Synthesis
To prepare a Drug-Linker Compound of the present invention, the
Drug is reacted with a reactive site on the Linker. In general, the
Linker can have the structure:
##STR00034## when both a Spacer unit (--Y--) and a Stretcher unit
(-A-) are present. Alternately, the Linker can have the
structure:
##STR00035## when the Spacer unit (--Y--) is absent.
The Linker can also have the structure:
##STR00036## when both the Stretcher unit (-A-) and the Spacer unit
(--Y--) are absent.
The Linker can also have the structure:
##STR00037## when both the Amino Acid unit (W) and the Spacer Unit
(Y) are absent.
In general, a suitable Linker has an Amino Acid unit linked to an
optional Stretcher Unit and an optional Spacer Unit. Reactive Site
1 is present at the terminus of the Spacer and Reactive site 2 is
present at the terminus of the Stretcher. If a Spacer unit is not
present, then Reactive site 1 is present at the C-terminus of the
Amino Acid unit.
In an exemplary embodiment of the invention, Reactive Site No. 1 is
reactive to a nitrogen atom of the Drug, and Reactive Site No. 2 is
reactive to a sulfhydryl group on the Ligand. Reactive Sites 1 and
2 can be reactive to different functional groups.
In one aspect of the invention, Reactive Site No. 1 is
##STR00038##
In another aspect of the invention, Reactive Site No. 1 is
##STR00039##
In still another aspect of the invention, Reactive Site No. 1 is a
p-nitrophenyl carbonate having the formula
##STR00040##
In one aspect of the invention, Reactive Site No. 2 is a
thiol-accepting group. Suitable thiol-accepting groups include
haloacetamide groups having the formula
##STR00041##
wherein X represents a leaving group, preferably O-mesyl, O-tosyl,
-Cl, -Br, or -I; or a maleimide group having the formula
##STR00042##
Useful Linkers can be obtained via commercial sources, such as
Molecular Biosciences Inc. (Boulder, Colo.), or prepared as
summarized in FIGS. 28-30.
In FIG. 28 X is --CH.sub.2-- or --CH.sub.2OCH.sub.2--; and n is an
integer ranging either from 0-10 when X is --CH.sub.2--; or 1-10
when X is --CH.sub.2OCH.sub.2--.
The method shown in FIG. 29 combines maleimide with a glycol under
Mitsunobu conditions to make a polyethylene glycol maleimide
Stretcher (see for example, Walker, M. A. J. Org. Chem. 1995, 60,
5352-5), followed by installation of a p-nitrophenyl carbonate
Reactive Site group.
In FIG. 29 E is --CH.sub.2-- or --CH.sub.2OCH.sub.2--; and e is an
integer ranging from 0-8;
Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can
be prepared as described by Frisch, et al., Bioconjugate Chem.
1996, 7, 180-186.
FIG. 30 illustrates a general synthesis of an illustrative Linker
unit containing a maleimide Stretcher group and optionally a
p-aminobenzyl ether self-immolative Spacer.
In FIG. 30 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; m is an integer ranging from
0-4; and n is an integer ranging from 0-10.
Useful Stretchers may be incorporated into a Linker using the
commercially available intermediates from Molecular Biosciences
(Boulder, Colo.) described below by utilizing known techniques of
organic synthesis.
Stretchers of formula (IIIa) can be introduced into a Linker by
reacting the following intermediates with the N-terminus of an
Amino Acid unit as depicted in FIGS. 31 and 32:
##STR00043##
where n is an integer ranging from 1-10 and T is --H or
--SO.sub.3Na;
##STR00044##
where n is an integer ranging from 0-3;
##STR00045##
Stretcher units of formula (IIIb) can be introduced into a Linker
by reacting the following intermediates with the N-terminus of an
Amino Acid unit:
##STR00046##
Stretcher units of formula (IV) can be introduced into a Linker by
reacting the following intermediates with the N-terminus of an
Amino Acid unit:
##STR00047##
Stretcher units of formula (Va) can be introduced into a Linker by
reacting the following intermediates with the N-terminus of an
Amino Acid unit:
##STR00048##
Other useful Stretchers may be synthesized according to known
procedures. Aminooxy Stretchers of the formula shown below can be
prepared by treating alkyl halides with N-Boc-hydroxylamine
according to procedures described in Jones, D. S. et al.,
Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C. et al.,
Tetrahedron, 1967, 23(11), 4441-4447.
##STR00049## wherein --R.sup.17-- is selected from
--C.sub.1-C.sub.10 alkylene-, --C.sub.3-C.sub.8 carbocyclo-,
--O--(C.sub.1-C.sub.8 alkyl)-, -arylene-, --C.sub.1-C.sub.10
alkylene-arylene-, -arylene-C.sub.1-C.sub.10 alkylene-,
--C.sub.1-C.sub.10 alkylene-(C.sub.3-C.sub.8 carbocyclo)-,
--(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(C.sub.3-C.sub.8 heterocyclo)-, --(C.sub.3-C.sub.8
heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10; Isothiocyanate Stretchers of the formula shown
below may be prepared from isothiocyanatocarboxylic acid chlorides
as described in Angew. Chem., 1975, 87(14):517.
##STR00050##
wherein --R.sup.17-- is as described herein.
FIG. 31 shows a method for obtaining of a val-cit dipeptide Linker
having a maleimide Stretcher and optionally a p-aminobenzyl
self-immolative Spacer.
In FIG. 31 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; and m is an integer ranging
from 0-4.
FIG. 32 illustrates the synthesis of a phe-lys(Mtr) dipeptide
Linker unit having a maleimide Stretcher unit and a p-aminobenzyl
self-immolative Spacer unit. Starting material AD (lys(Mtr)) is
commercially available (Bachem, Torrance, Calif.) or can be
prepared according to Dubowchik, et al. Tetrahedron Letters (1997)
38:5257-60.
In FIG. 32 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; and m is an integer ranging
from 0-4.
As shown in FIG. 33, a Linker can be reacted with an amino group of
a Drug Compound of Formula (Ib) to form a Drug-Linker Compound that
contains an amide or carbamate group, linking the Drug unit to the
Linker unit. When Reactive Site No. 1 is a carboxylic acid group,
as in Linker AJ, the coupling reaction can be performed using HATU
or PyBrop and an appropriate amine base, resulting in a Drug-Linker
Compound AK, containing an amide bond between the Drug unit and the
Linker unit. When Reactive Site No. 1 is a carbonate, as in Linker
AL, the Linker can be coupled to the Drug using HOBt in a mixture
of DMF/pyridine to provide a Drug-Linker Compound AM, containing a
carbamate bond between the Drug unit and the Linker unit
Alternately, when Reactive Site No. 1 is a good leaving group, such
as in Linker AN, the Linker can be coupled with an amine group of a
Drug via a nucleophilic substitution process to provide a
Drug-Linker Compound having an amine linkage (AO) between the Drug
unit and the Linker unit.
Illustrative methods useful for linking a Drug to a Ligand to form
a Drug-Linker Compound are depicted in FIG. 33 and are outlined in
General Procedures G-H.
General Procedure G: Amide Formation Using HATU.
A Drug (Ib) (1.0 eq.) and an N-protected Linker containing a
carboxylic acid Reactive site (1.0 eq.) are diluted with a suitable
organic solvent, such as dichloromethane, and the resulting
solution is treated with HATU (1.5 eq.) and an organic base,
preferably pyridine (1.5 eq.). The reaction mixture is allowed to
stir under an inert atmosphere, preferably argon, for 6 h, during
which time the reaction mixture is monitored using HPLC. The
reaction mixture is concentrated and the resulting residue is
purified using HPLC to yield the amide of formula AK.
Procedure H: Carbamate Formation Using HOBt.
A mixture of a Linker AL having a p-nitrophenyl carbonate Reactive
site (1.1 eq.) and Drug (Ib) (1.0 eq.) are diluted with an aprotic
organic solvent, such as DMF, to provide a solution having a
concentration of 50-100 mM, and the resulting solution is treated
with HOBt (2.0 eq.) and placed under an inert atmosphere,
preferably argon. The reaction mixture is allowed to stir for 15
min, then an organic base, such as pyridine (1/4 v/v), is added and
the reaction progress is monitored using HPLC. The Linker is
typically consumed within 16 h. The reaction mixture is then
concentrated in vacuo and the resulting residue is purified using,
for example, HPLC to yield the carbamate AM.
An alternate method of preparing Drug-Linker Compounds is outlined
in FIG. 34. Using the method of FIG. 34, the Drug is attached to a
partial Linker unit (ZA, for example), which does not have a
Stretcher unit attached. This provides intermediate AP, which has
an Amino Acid unit having an Fmoc-protected N-terminus. The Fmoc
group is then removed and the resulting amine intermediate AQ is
then attached to a Stretcher unit via a coupling reaction catalyzed
using PyBrop or DEPC. The construction of Drug-Linker Compounds
containing either a bromoacetamide Stretcher AR or a PEG maleimide
Stretcher AS is illustrated in FIG. 34.
In FIG. 34 Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -halogen, -nitro or -cyano; and m is an integer ranging
from 0-4.
Methodology useful for the preparation of a Linker unit containing
a branched spacer is shown in FIG. 35.
FIG. 35 illustrates the synthesis of a val-cit dipeptide linker
having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene
(BHMS) unit. The synthesis of the BHMS intermediate (AW) has been
improved from previous literature procedures (see International
Publication No, WO 9813059 to Firestone et al., and Crozet, M. P.;
Archaimbault, G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett.
(1985) 26:5133-5134) and utilizes as starting materials,
commercially available diethyl (4-nitrobenzyl)phosphonate (AT) and
commercially available 2,2-dimethyl-1,3-dioxan-5-one (AU). Linkers
AY and BA can be prepared from intermediate AW using the
methodology described in FIG. 29.
4.6.3 Dendritic Linkers
The linker may be a dendritic type linker for covalent attachment
of more than one drug moiety through a branching, multifunctional
linker moiety to a Ligand, such as but not limited to an antibody
(Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters
12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal
Chemistry 11:1761-1768). Dendritic linkers can increase the molar
ratio of drug to antibody, i.e. loading, which is related to the
potency of the Drug-Linker-Ligand Conjugate. Thus, where a cysteine
engineered antibody bears only one reactive cytsteine thiol group,
a multitude of drug moieties may be attached through a dendritic
linker.
The following exemplary embodiments of dendritic linker reagents
allow up to nine nucleophilic drug moiety reagents to be conjugated
by reaction with the chloroethyl nitrogen mustard functional
groups:
##STR00051## 4.6.4 Conjugation of Drug Moieties to Antibodies
FIG. 36 illustrates methodology useful for making
Drug-Linker-Ligand conjugates having about 2 to about 4 drugs per
antibody. An antibody is treated with a reducing agent, such as
dithiothreitol (DTT) to reduce some or all of the cysteine
disulfide residues to form highly nucleophilic cysteine thiol
groups (--CH.sub.2SH). The partially reduced antibody thus reacts
with drug-linker compounds, or linker reagents, with electrophilic
functional groups such as maleimide or a-halo carbonyl, according
to the conjugation method at page 766 of Klussman, et al. (2004),
Bioconjugate Chemistry 15(4):765-773.
For example, an antibody, e.g., AC10, dissolved in 500 mM sodium
borate and 500 mM sodium chloride at pH 8.0 is treated with an
excess of 100 mM dithiothreitol (DTT). After incubation at
37.degree. C. for about 30 minutes, the buffer is exchanged by
elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA.
The thiol/Ab value is checked by determining the reduced antibody
concentration from the absorbance at 280 nm of the solution and the
thiol concentration by reaction with DTNB (Aldrich, Milwaukee,
Wis.) and determination of the absorbance at 412 nm. The reduced
antibody dissolved in PBS is chilled on ice. The drug linker, e.g.,
MC-val-cit-PAB-MMAE in DMSO, dissolved in acetonitrile and water at
known concentration, is added to the chilled reduced antibody in
PBS. After about one hour, an excess of maleimide is added to
quench the reaction and cap any unreacted antibody thiol groups.
The reaction mixture is concentrated by centrifugal ultrafiltration
and the ADC, e.g., AC10-MC-vc-PAB-MMAE, is purified and desalted by
elution through G25 resin in PBS, filtered through 0.2 .mu.m
filters under sterile conditions, and frozen for storage.
A variety of antibody drug conjugates (ADC) were prepared, with a
variety of linkers, and the drug moieties, MMAE and MMAF. The
following table is an exemplary group of ADC which were prepared
following the protocol of Example 27, and characterized by HPLC and
drug loading assay.
TABLE-US-00041 isolated Target amount drug/Ab (antigen) ADC (mg)
ratio 0772P 16E12-MC-vc-PAB-MMAE 1.75 4 0772P 11D10-MC-vc-PAB-MMAE
46.8 4.4 0772P 11D10-MC-vc-PAB-MMAF 54.5 3.8 Brevican
Brevican-MC-MMAF 2 6 Brevican Brevican-MC-vc-MMAF 2 6 Brevican
Brevican-MC-vc-PAB-MMAF 1.4 6 CD21 CD21-MC-vc-PAB-MMAE 38.1 4.3
CD21 CD21-MC-vc-PAB-MMAF 43 4.1 CRIPTO 11F4-MC-vc-PAB-MMAF 6 4.8
CRIPTO 25G8-MC-vc-PAB-MMAF 7.4 4.7 E16 12G12-MC-vc-PAB-MMAE 2.3 4.6
E16 3B5-MC-vc-PAB-MMAE 2.9 4.6 E16 12B9-MC-vc-PAB-MMAE 1.4 3.8 E16
12B9-MC-vc-PAB-MMAE 5.1 4 E16 12G12-MC-vc-PAB-MMAE 3 4.6 E16
3B5-MC-vc-PAB-MMAE 4.8 4.1 E16 3B5-MC-vc-PAB-MMAF 24.7 4.4 EphB2R
2H9-MC-vc-PAB-MMAE 29.9 7.1 EphB2R 2H9-MC-fk-PAB-MMAE 25 7.5 EphB2R
2H9-MC-vc-PAB-MMAE 175 4.1 EphB2R 2H9-MC-vc-PAB-MMAF 150 3.8 EphB2R
2H9-MC-vc-PAB-MMAF 120 3.7 EphB2R 2H9-MC-vc-PAB-MMAE 10.7 4.4
IL-20Ra IL20Ra-fk-MMAE 26 6.7 IL-20Ra IL20Ra-vc-MMAE 27 7.3 EphB2
IL8-MC-vc-PAB-MMAE 251 3.7 MDP MDP-vc-MMAE 32 MPF 19C3-vc-MMAE 1.44
6.5 MPF 7D9-vc-MMAE 4.3 3.8 MPF 19C3-vc-MMAE 7.9 3 MPF
7D9-MC-vc-PAB-MMAF 5 4.3 Napi3b 10H1-vc-MMAE 4.5 4.6 Napi3b
4C9-vc-MMAE 3.0 5.4 Napi3b 10H1-vc-MMAE 4.5 4.8 Napi3b 10H1-vc-MMAF
6.5 4 NCA 3E6-MC-fk-PAB-MMAE 49.6 5.4 NCA 3E6-MC-vc-PAB-MMAE 56.2
6.4 PSCA PSCA-fk-MMAE 51.7 8.9 PSCA PSCA-vc-MMAE 61.1 8.6 Napi3b
10H1-MC-vc-PAB-MMAE 75 4.2 Napi3b 10H1-MC-vc-PAB-MMAF 95 4.4 Napi3b
10H1-MC-MMAF 92 4 EphB2R 2H9-MC-vc-PAB-MMAE 79 5 EphB2R 2H9-MC-MMAF
92 4.9 0772P 11D10(Fc chimera)-MC-vc-PAB- 79 4.3 MMAE 0772P
11D10(Fc chimera)-MC-vc-PAB- 70 4.5 MMAF 0772P 11D10(Fc
chimera)-MC-MMAF 23 4.5 Brevican 6D2-MC-vc-PAB-MMAF 0.3 4.5
Brevican 6D2-MC-MMAF 0.36 4.5 EphB2R 2H9(Fc chimera)-MC-vc-PAB-MMAE
1983 4.3 E16 12B9-MC-vc-PAB-MMAE 14.1 4.6 E16 12B9-MC-vc-PAB-MMAF
16.4 4.5 E16 12G12-MC-vc-PAB-MMAE 10.5 4.1 E16 12G12-MC-vc-PAB-MMAF
10.2 3.8 E16 3B5-MC-vc-PAB-MMAE 58.6 3.8 E16 3B5-MC-vc-PAB-MMAF 8
3.1 0772P 11D10(Fc chimera)-MC-vc-PAB- 340 3.9 MMAE Steap1
(Steap1-92)-MC-vc-PAB-MMAE 3.5 4 Steap1 (Steap1-92)-MC-vc-PAB-MMAF
4.7 4 Steap1 (Steap1-120)-MC-vc-PAB- 2 4 MMAE Steap1
(Steap1-120)-MC-vc-PAB-MMAF 2.3 4 E16 3B5-MC-vc-PAB-MMAF 52.2
4.5
4.7 Compositions and Methods of Administration
In other embodiments, described is a composition including an
effective amount of an Exemplary Compound and/or Exemplary
Conjugate and a pharmaceutically acceptable carrier or vehicle. For
convenience, the Drug units and Drug-Linker Compounds can be
referred to as Exemplary Compounds, while Drug-Ligand Conjugates
and Drug-Linker-Ligand Conjugates can be referred to as Exemplary
Conjugates. The compositions are suitable for veterinary or human
administration.
The present compositions can be in any form that allows for the
composition to be administered to a patient. For example, the
composition can be in the form of a solid, liquid or gas (aerosol).
Typical routes of administration include, without limitation, oral,
topical, parenteral, sublingual, rectal, vaginal, ocular,
intra-tumor, and intranasal. Parenteral administration includes
subcutaneous injections, intravenous, intramuscular, intrasternal
injection or infusion techniques. In one aspect, the compositions
are administered parenterally. In yet another aspect, the Exemplary
Compounds and/or the Exemplary Conjugates or compositions are
administered intravenously.
Pharmaceutical compositions can be formulated so as to allow an
Exemplary Compound and/or Exemplary Conjugate to be bioavailable
upon administration of the composition to a patient. Compositions
can take the form of one or more dosage units, where for example, a
tablet can be a single dosage unit, and a container of an Exemplary
Compound and/or Exemplary Conjugate in aerosol form can hold a
plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be
non-toxic in the amounts used. It will be evident to those of
ordinary skill in the art that the optimal dosage of the active
ingredient(s) in the pharmaceutical composition will depend on a
variety of factors. Relevant factors include, without limitation,
the type of animal (e.g., human), the particular form of the
Exemplary Compound or Exemplary Conjugate, the manner of
administration, and the composition employed.
The pharmaceutically acceptable carrier or vehicle can be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) can be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) can be gaseous or particulate, so as to
provide an aerosol composition useful in, e.g., inhalatory
administration.
When intended for oral administration, the composition is
preferably in solid or liquid form, where semi-solid, semi-liquid,
suspension and gel forms are included within the forms considered
herein as either solid or liquid.
As a solid composition for oral administration, the composition can
be formulated into a powder, granule, compressed tablet, pill,
capsule, chewing gum, wafer or the like form. Such a solid
composition typically contains one or more inert diluents. In
addition, one or more of the following can be present: binders such
as carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins, disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon
dioxide; sweetening agents such as sucrose or saccharin, a
flavoring agent such as peppermint, methyl salicylate or orange
flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin
capsule, it can contain, in addition to materials of the above
type, a liquid carrier such as polyethylene glycol, cyclodextrin or
a fatty oil.
The composition can be in the form of a liquid, e.g., an elixir,
syrup, solution, emulsion or suspension. The liquid can be useful
for oral administration or for delivery by injection. When intended
for oral administration, a composition can comprise one or more of
a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition for administration by injection, one or
more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent can
also be included.
The liquid compositions, whether they are solutions, suspensions or
other like form, can also include one or more of the following:
sterile diluents such as water for injection, saline solution,
preferably physiological saline, Ringer's solution, isotonic sodium
chloride, fixed oils such as synthetic mono or digylcerides which
can serve as the solvent or suspending medium, polyethylene
glycols, glycerin, cyclodextrin, propylene glycol or other
solvents; antibacterial agents such as benzyl alcohol or methyl
paraben; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid; buffers
such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. A
parenteral composition can be enclosed in ampoule, a disposable
syringe or a multiple-dose vial made of glass, plastic or other
material. Physiological saline is an exemplary adjuvant. An
injectable composition is preferably sterile.
The amount of the Exemplary Compound and/or Exemplary Conjugate
that is effective in the treatment of a particular disorder or
condition will depend on the nature of the disorder or condition,
and can be determined by standard clinical techniques. In addition,
in vitro or in vivo assays can optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in
the compositions will also depend on the route of administration,
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
patient's circumstances.
The compositions comprise an effective amount of an Exemplary
Compound and/or Exemplary Conjugate such that a suitable dosage
will be obtained. Typically, this amount is at least about 0.01% of
an Exemplary Compound and/or Exemplary Conjugate by weight of the
composition. When intended for oral administration, this amount can
be varied to range from about 0.1% to about 80% by weight of the
composition. In one aspect, oral compositions can comprise from
about 4% to about 50% of the Exemplary Compound and/or Exemplary
Conjugate by weight of the composition. In yet another aspect,
present compositions are prepared so that a parenteral dosage unit
contains from about 0.01% to about 2% by weight of the Exemplary
Compound and/or Exemplary Conjugate.
For intravenous administration, the composition can comprise from
about 0.01 to about 100 mg of an Exemplary Compound and/or
Exemplary Conjugate per kg of the animal's body weight. In one
aspect, the composition can include from about 1 to about 100 mg of
an Exemplary Compound and/or Exemplary Conjugate per kg of the
animal's body weight. In another aspect, the amount administered
will be in the range from about 0.1 to about 25 mg/kg of body
weight of the Exemplary Compound and/or Exemplary Conjugate.
Generally, the dosage of an Exemplary Compound and/or Exemplary
Conjugate administered to a patient is typically about 0.01 mg/kg
to about 2000 mg/kg of the animal's body weight. In one aspect, the
dosage administered to a patient is between about 0.01 mg/kg to
about 10 mg/kg of the animal's body weight, in another aspect, the
dosage administered to a patient is between about 0.1 mg/kg and
about 250 mg/kg of the animal's body weight, in yet another aspect,
the dosage administered to a patient is between about 0.1 mg/kg and
about 20 mg/kg of the animal's body weight, in yet another aspect
the dosage administered is between about 0.1 mg/kg to about 10
mg/kg of the animal's body weight, and in yet another aspect, the
dosage administered is between about 1 mg/kg to about 10 mg/kg of
the animal's body weight.
The Exemplary Compounds and/or Exemplary Conjugate or compositions
can be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.). Administration can be systemic or local. Various
delivery systems are known, e.g., encapsulation in liposomes,
microparticles, microcapsules, capsules, etc., and can be used to
administer an Exemplary Compound and/or Exemplary Conjugate or
composition. In certain embodiments, more than one Exemplary
Compound and/or Exemplary Conjugate or composition is administered
to a patient.
In specific embodiments, it can be desirable to administer one or
more Exemplary Compounds and/or Exemplary Conjugate or compositions
locally to the area in need of treatment. This can be achieved, for
example, and not by way of limitation, by local infusion during
surgery; topical application, e.g., in conjunction with a wound
dressing after surgery; by injection; by means of a catheter; by
means of a suppository; or by means of an implant, the implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. In one
embodiment, administration can be by direct injection at the site
(or former site) of a cancer, tumor or neoplastic or pre-neoplastic
tissue. In another embodiment, administration can be by direct
injection at the site (or former site) of a manifestation of an
autoimmune disease.
In certain embodiments, it can be desirable to introduce one or
more Exemplary Compounds and/or Exemplary Conjugate or compositions
into the central nervous system by any suitable route, including
intraventricular and intrathecal injection. Intraventricular
injection can be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent,
or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant.
In yet another embodiment, the Exemplary Compounds and/or Exemplary
Conjugate or compositions can be delivered in a controlled release
system, such as but not limited to, a pump or various polymeric
materials can be used. In yet another embodiment, a
controlled-release system can be placed in proximity of the target
of the Exemplary Compounds and/or Exemplary Conjugate or
compositions, e.g., the brain, thus requiring only a fraction of
the systemic dose (see, e.g., Goodson, in Medical Applications of
Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other
controlled-release systems discussed in the review by Langer
(Science 249:1527-1533 (1990)) can be used.
The term "carrier" refers to a diluent, adjuvant or excipient, with
which an Exemplary Compound and/or Exemplary Conjugate is
administered. Such pharmaceutical carriers can be liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. The carriers can be saline, gum acacia,
gelatin, starch paste, talc, keratin, colloidal silica, urea, and
the like. In addition, auxiliary, stabilizing, thickening,
lubricating and coloring agents can be used. In one embodiment,
when administered to a patient, the Exemplary Compound and/or
Exemplary Conjugate or compositions and pharmaceutically acceptable
carriers are sterile. Water is an exemplary carrier when the
Exemplary Compounds and/or Exemplary Conjugates are administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers also include
excipients such as starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The present
compositions, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, aerosols, sprays, suspensions, or any
other form suitable for use. Other examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
In an embodiment, the Exemplary Compounds and/or Exemplary
Conjugates are formulated in accordance with routine procedures as
a pharmaceutical composition adapted for intravenous administration
to animals, particularly human beings. Typically, the carriers or
vehicles for intravenous administration are sterile isotonic
aqueous buffer solutions. Where necessary, the compositions can
also include a solubilizing agent. Compositions for intravenous
administration can optionally comprise a local anesthetic such as
lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
an Exemplary Compound and/or Exemplary Conjugate is to be
administered by infusion, it can be dispensed, for example, with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the Exemplary Compound and/or Exemplary Conjugate is
administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients can be
mixed prior to administration.
Compositions for oral delivery can be in the form of tablets,
lozenges, aqueous or oily suspensions, granules, powders,
emulsions, capsules, syrups, or elixirs, for example. Orally
administered compositions can contain one or more optionally
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents such as peppermint, oil of
wintergreen, or cherry; coloring agents; and preserving agents, to
provide a pharmaceutically palatable preparation. Moreover, where
in tablet or pill form, the compositions can be coated to delay
disintegration and absorption in the gastrointestinal tract thereby
providing a sustained action over an extended period of time.
Selectively permeable membranes surrounding an osmotically active
driving compound are also suitable for orally administered
compounds. In these later platforms, fluid from the environment
surrounding the capsule is imbibed by the driving compound, which
swells to displace the agent or agent composition through an
aperture. These delivery platforms can provide an essentially zero
order delivery profile as opposed to the spiked profiles of
immediate release formulations. A time-delay material such as
glycerol monostearate or glycerol stearate can also be used.
The compositions can be intended for topical administration, in
which case the carrier may be in the form of a solution, emulsion,
ointment or gel base. If intended for transdermal administration,
the composition can be in the form of a transdermal patch or an
iontophoresis device. Topical formulations can comprise a
concentration of an Exemplary Compound and/or Exemplary Conjugate
of from about 0.05% to about 50% w/v (weight per unit volume of
composition), in another aspect, from 0.1% to 10% w/v.
The composition can be intended for rectal administration, in the
form, e.g., of a suppository which will melt in the rectum and
release the Exemplary Compound and/or Exemplary Conjugate.
The composition can include various materials that modify the
physical form of a solid or liquid dosage unit. For example, the
composition can include materials that form a coating shell around
the active ingredients. The materials that form the coating shell
are typically inert, and can be selected from, for example, sugar,
shellac, and other enteric coating agents. Alternatively, the
active ingredients can be encased in a gelatin capsule.
The compositions can consist of gaseous dosage units, e.g., it can
be in the form of an aerosol. The term aerosol is used to denote a
variety of systems ranging from those of colloidal nature to
systems consisting of pressurized packages. Delivery can be by a
liquefied or compressed gas or by a suitable pump system that
dispenses the active ingredients.
Whether in solid, liquid or gaseous form, the present compositions
can include a pharmacological agent used in the treatment of
cancer, an autoimmune disease or an infectious disease.
4.8 Therapeutic Uses of the Exemplary Conjugates
The Exemplary Compounds and/or Exemplary Conjugates are useful for
treating cancer, an autoimmune disease or an infectious disease in
a patient.
4.8.1 Treatment of Cancer
The Exemplary Compounds and/or Exemplary Conjugates are useful for
inhibiting the multiplication of a tumor cell or cancer cell,
causing apoptosis in a tumor or cancer cell, or for treating cancer
in a patient. The Exemplary Compounds and/or Exemplary Conjugates
can be used accordingly in a variety of settings for the treatment
of animal cancers. The Drug-Linker-Ligand Conjugates can be used to
deliver a Drug or Drug unit to a tumor cell or cancer cell. Without
being bound by theory, in one embodiment, the Ligand unit of an
Exemplary Conjugate binds to or associates with a cancer-cell or a
tumor-cell-associated antigen, and the Exemplary Conjugate can be
taken up inside a tumor cell or cancer cell through
receptor-mediated endocytosis. The antigen can be attached to a
tumor cell or cancer cell or can be an extracellular matrix protein
associated with the tumor cell or cancer cell. Once inside the
cell, one or more specific peptide sequences within the Linker unit
are hydrolytically cleaved by one or more tumor-cell or
cancer-cell-associated proteases, resulting in release of a Drug or
a Drug-Linker Compound. The released Drug or Drug-Linker Compound
is then free to migrate within the cell and induce cytotoxic or
cytostatic activities. In an alternative embodiment, the Drug or
Drug unit is cleaved from the Exemplary Conjugate outside the tumor
cell or cancer cell, and the Drug or Drug-Linker Compound
subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the tumor cell or
cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or
cancer cell antigen which is on the surface of the tumor cell or
cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or
cancer cell antigen which is an extracellular matrix protein
associated with the tumor cell or cancer cell.
The specificity of the Ligand unit for a particular tumor cell or
cancer cell can be important for determining those tumors or
cancers that are most effectively treated. For example, Exemplary
Conjugates having a BR96 Ligand unit can be useful for treating
antigen positive carcinomas including those of the lung, breast,
colon, ovaries, and pancreas. Exemplary Conjugates having an
Anti-CD30 or an anti-CD40 Ligand unit can be useful for treating
hematologic malignancies.
Other particular types of cancers that can be treated with
Exemplary Conjugates include, but are not limited to, those
disclosed in Table 3.
TABLE-US-00042 TABLE 3 Solid tumors, including but not limited to:
fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic
sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma
lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor
leiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer
kidney cancer pancreatic cancer bone cancer breast cancer ovarian
cancer prostate cancer esophogeal cancer stomach cancer oral cancer
nasal cancer throat cancer squamous cell carcinoma basal cell
carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland
carcinoma papillary carcinoma papillary adenocarcinomas
cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal
cell carcinoma hepatoma bile duct carcinoma choriocarcinoma
seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine
cancer testicular cancer small cell lung carcinoma bladder
carcinoma lung cancer epithelial carcinoma glioma glioblastoma
multiforme astrocytoma medulloblastoma craniopharyngioma ependymoma
pinealoma hemangioblastoma acoustic neuroma oligodendroglioma
meningioma skin cancer melanoma neuroblastoma retinoblastoma
blood-borne cancers, including but not limited to: acute
lymphoblastic leukemia "ALL" acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia acute myeloblastic leukemia
"AML" acute promyelocytic leukemia "APL" acute monoblastic leukemia
acute erythroleukemic leukemia acute megakaryoblastic leukemia
acute myelomonocytic leukemia acute nonlymphocyctic leukemia acute
undifferentiated leukemia chronic myelocytic leukemia "CML" chronic
lymphocytic leukemia "CLL" hairy cell leukemia multiple myeloma
acute and chronic leukemias: lymphoblastic myelogenous lymphocytic
myelocytic leukemias Lymphomas: Hodgkin's disease non-Hodgkin's
Lymphoma Multiple myeloma Waldenstrom's macroglobulinemia Heavy
chain disease Polycythemia vera
The Exemplary Conjugates provide conjugation-specific tumor or
cancer targeting, thus reducing general toxicity of these
compounds. The Linker units stabilize the Exemplary Conjugates in
blood, yet are cleavable by tumor-specific proteases within the
cell, liberating a Drug.
4.8.2 Multi-Modality Therapy for Cancer
Cancers, including, but not limited to, a tumor, metastasis, or
other disease or disorder characterized by uncontrolled cell
growth, can be treated or prevented by administration of an
Exemplary Conjugate and/or an Exemplary Compound.
In other embodiments, methods for treating or preventing cancer are
provided, including administering to a patient in need thereof an
effective amount of an Exemplary Conjugate and a chemotherapeutic
agent. In one embodiment the chemotherapeutic agent is that with
which treatment of the cancer has not been found to be refractory.
In another embodiment, the chemotherapeutic agent is that with
which the treatment of cancer has been found to be refractory. The
Exemplary Conjugates can be administered to a patient that has also
undergone surgery as treatment for the cancer.
In one embodiment, the additional method of treatment is radiation
therapy.
In a specific embodiment, the Exemplary Conjugate is administered
concurrently with the chemotherapeutic agent or with radiation
therapy. In another specific embodiment, the chemotherapeutic agent
or radiation therapy is administered prior or subsequent to
administration of an Exemplary Conjugates, in one aspect at least
an hour, five hours, 12 hours, a day, a week, a month, in further
aspects several months (e.g., up to three months), prior or
subsequent to administration of an Exemplary Conjugate.
A chemotherapeutic agent can be administered over a series of
sessions. Any one or a combination of the chemotherapeutic agents
listed in Table 4 can be administered. With respect to radiation,
any radiation therapy protocol can be used depending upon the type
of cancer to be treated. For example, but not by way of limitation,
x-ray radiation can be administered; in particular, high-energy
megavoltage (radiation of greater that 1 MeV energy) can be used
for deep tumors, and electron beam and orthovoltage x-ray radiation
can be used for skin cancers. Gamma-ray emitting radioisotopes,
such as radioactive isotopes of radium, cobalt and other elements,
can also be administered.
Additionally, methods of treatment of cancer with an Exemplary
Compound and/or Exemplary Conjugate are provided as an alternative
to chemotherapy or radiation therapy where the chemotherapy or the
radiation therapy has proven or can prove too toxic, e.g., results
in unacceptable or unbearable side effects, for the subject being
treated. The animal being treated can, optionally, be treated with
another cancer treatment such as surgery, radiation therapy or
chemotherapy, depending on which treatment is found to be
acceptable or bearable.
The Exemplary Compounds and/or Exemplary Conjugates can also be
used in an in vitro or ex vivo fashion, such as for the treatment
of certain cancers, including, but not limited to leukemias and
lymphomas, such treatment involving autologous stem cell
transplants. This can involve a multi-step process in which the
animal's autologous hematopoietic stem cells are harvested and
purged of all cancer cells, the animal's remaining bone-marrow cell
population is then eradicated via the administration of a high dose
of an Exemplary Compound and/or Exemplary Conjugate with or without
accompanying high dose radiation therapy, and the stem cell graft
is infused back into the animal. Supportive care is then provided
while bone marrow function is restored and the animal recovers.
4.8.3 Multi-Drug Therapy for Cancer
Methods for treating cancer including administering to a patient in
need thereof an effective amount of an Exemplary Conjugate and
another therapeutic agent that is an anti-cancer agent are
disclosed. Suitable anticancer agents include, but are not limited
to, methotrexate, taxol, L-asparaginase, mercaptopurine,
thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine,
procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide,
5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin,
doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,
mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,
paclitaxel, and docetaxel. In one aspect, the anti-cancer agent
includes, but is not limited to, a drug listed in Table 4.
TABLE-US-00043 TABLE 4 Alkylating agents Nitrogen mustards:
cyclophosphamide ifosfamide trofosfamide chlorambucil melphalan
Nitrosoureas: carmustine (BCNU) lomustine (CCNU) Alkylsulphonates
busulfan treosulfan Triazenes: decarbazine Platinum containing
compounds: cisplatin carboplatin Plant Alkaloids Vinca alkaloids:
vincristine vinblastine vindesine vinorelbine Taxoids: paclitaxel
docetaxol DNA Topoisomerase Inhibitors Epipodophyllins: etoposide
teniposide topotecan 9-aminocamptothecin camptothecin crisnatol
mitomycins: mitomycin C Anti-metabolites Anti-folates: DHFR
inhibitors: methotrexate trimetrexate IMP dehydrogenase Inhibitors:
mycophenolic acid tiazofurin ribavirin EICAR Ribonucleotide
reductase Inhibitors: hydroxyurea deferoxamine Pyrimidine analogs:
Uracil analogs 5-Fluorouracil floxuridine doxifluridine ratitrexed
Cytosine analogs cytarabine (ara C) cytosine arabinoside
fludarabine Purine analogs: mercaptopurine thioguanine Hormonal
therapies: Receptor antagonists: Anti-estrogen tamoxifen raloxifene
megestrol LHRH agonists: goscrclin leuprolide acetate
Anti-androgens: flutamide bicalutamide Retinoids/Deltoids Vitamin
D3 analogs: EB 1089 CB 1093 KH 1060 Photodynamic therapies:
vertoporfin (BPD-MA) phthalocyanine photosensitizer Pc4
demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines: Interferon-.alpha.
Interferon-.gamma. tumor necrosis factor Others: Gemcitabine
Velcade Revamid Thalamid Isoprenylation inhibitors: Lovastatin
Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell
cycle inhibitors: staurosporine Actinomycins: Actinomycin D
dactinomycin Bleomycins: bleomycin A2 bleomycin B2 peplomycin
Anthracyclines: daunorubicin Doxorubicin (adriamycin) idarubicin
epirubicin pirarubicin zorubicin mtoxantrone MDR inhibitors:
verapamil Ca.sup.2+ATPase inhibitors: thapsigargin
4.8.4 Treatment of Autoimmune Diseases
The Exemplary Conjugates are useful for killing or inhibiting the
replication of a cell that produces an autoimmune disease or for
treating an autoimmune disease. The Exemplary Conjugates can be
used accordingly in a variety of settings for the treatment of an
autoimmune disease in a patient. The Drug-Linker-Ligand Conjugates
can be used to deliver a Drug to a target cell. Without being bound
by theory, in one embodiment, the Drug-Linker-Ligand Conjugate
associates with an antigen on the surface of a target cell, and the
Exemplary Conjugate is then taken up inside a target-cell through
receptor-mediated endocytosis. Once inside the cell, one or more
specific peptide sequences within the Linker unit are enzymatically
or hydrolytically cleaved, resulting in release of a Drug. The
released Drug is then free to migrate in the cytosol and induce
cytotoxic or cytostatic activities. In an alternative embodiment,
the Drug is cleaved from the Exemplary Conjugate outside the target
cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to an autoimmune antigen.
In one aspect, the antigen is on the surface of a cell involved in
an autoimmune condition.
In another embodiment, the Ligand unit binds to an autoimmune
antigen which is on the surface of a cell.
In one embodiment, the Ligand binds to activated lymphocytes that
are associated with the autoimmune disease state.
In a further embodiment, the Exemplary Conjugates kill or inhibit
the multiplication of cells that produce an autoimmune antibody
associated with a particular autoimmune disease.
Particular types of autoimmune diseases that can be treated with
the Exemplary Conjugates include, but are not limited to, Th2
lymphocyte related disorders (e.g., atopic dermatitis, atopic
asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,
systemic sclerosis, and graft versus host disease); Th1
lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple
sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,
Grave's disease, primary biliary cirrhosis, Wegener's
granulomatosis, and tuberculosis); activated B lymphocyte-related
disorders (e.g., systemic lupus erythematosus, Goodpasture's
syndrome, rheumatoid arthritis, and type I diabetes); and those
disclosed in Table 5.
TABLE-US-00044 TABLE 5 Active Chronic Hepatitis Addison's Disease
Allergic Alveolitis Allergic Reaction Allergic Rhinitis Alport's
Syndrome Anaphlaxis Ankylosing Spondylitis Anti-phosholipid
Syndrome Arthritis Ascariasis Aspergillosis Atopic Allergy Atropic
Dermatitis Atropic Rhinitis Behcet's Disease Bird-Fancier's Lung
Bronchial Asthma Caplan's Syndrome Cardiomyopathy Celiac Disease
Chagas' Disease Chronic Glomerulonephritis Cogan's Syndrome Cold
Agglutinin Disease Congenital Rubella Infection CREST Syndrome
Crohn's Disease Cryoglobulinemia Cushing's Syndrome Dermatomyositis
Discoid Lupus Dressler's Syndrome Eaton-Lambert Syndrome Echovirus
Infection Encephalomyelitis Endocrine opthalmopathy Epstein-Barr
Virus Infection Equine Heaves Erythematosis Evan's Syndrome Felty's
Syndrome Fibromyalgia Fuch's Cyclitis Gastric Atrophy
Gastrointestinal Allergy Giant Cell Arteritis Glomerulonephritis
Goodpasture's Syndrome Graft v. Host Disease Graves' Disease
Guillain-Barre Disease Hashimoto's Thyroiditis Hemolytic Anemia
Henoch-Schonlein Purpura Idiopathic Adrenal Atrophy Idiopathic
Pulmonary Fibritis IgA Nephropathy Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus Juvenile Arthritis Juvenile
Diabetes Mellitus (Type I) Lambert-Eaton Syndrome Laminitis Lichen
Planus Lupoid Hepatitis Lupus Lymphopenia Meniere's Disease Mixed
Connective Tissue Disease Multiple Sclerosis Myasthenia Gravis
Pernicious Anemia Polyglandular Syndromes Presenile Dementia
Primary Agammaglobulinemia Primary Biliary Cirrhosis Psoriasis
Psoriatic Arthritis Raynauds Phenomenon Recurrent Abortion Reiter's
Syndrome Rheumatic Fever Rheumatoid Arthritis Sampter's Syndrome
Schistosomiasis Schmidt's Syndrome Scleroderma Shulman's Syndrome
Sjorgen's Syndrome Stiff-Man Syndrome Sympathetic Ophthalmia
Systemic Lupus Erythematosis Takayasu's Arteritis Temporal
Arteritis Thyroiditis Thrombocytopenia Thyrotoxicosis Toxic
Epidermal Necrolysis Type B Insulin Resistance Type I Diabetes
Mellitus Ulcerative Colitis Uveitis Vitiligo Waldenstrom's
Macroglobulemia Wegener's Granulomatosis
4.8.5 Multi-Drug Therapy of Autoimmune Diseases
Methods for treating an autoimmune disease are also disclosed
including administering to a patient in need thereof an effective
amount of an Exemplary Conjugate and another therapeutic agent
known for the treatment of an autoimmune disease. In one
embodiment, the anti-autoimmune disease agent includes, but is not
limited to, agents listed in Table 6.
TABLE-US-00045 TABLE 6 cyclosporine cyclosporine A mycophenylate
mofetil sirolimus tacrolimus enanercept prednisone azathioprine
methotrexate cyclophosphamide prednisone aminocaproic acid
chloroquine hydroxychloroquine hydrocortisone dexamethasone
chlorambucil DHEA danazol bromocriptine meloxicam infliximab
4.8.6 Treatment of Infectious Diseases
The Exemplary Conjugates are useful for killing or inhibiting the
multiplication of a cell that produces an infectious disease or for
treating an infectious disease. The Exemplary Conjugates can be
used accordingly in a variety of settings for the treatment of an
infectious disease in a patient. The Drug-Linker-Ligand Conjugates
can be used to deliver a Drug to a target cell. In one embodiment,
the Ligand unit binds to the infectious disease cell.
In one embodiment, the Conjugates kill or inhibit the
multiplication of cells that produce a particular infectious
disease.
Particular types of infectious diseases that can be treated with
the Exemplary Conjugates include, but are not limited to, those
disclosed in Table 7.
TABLE-US-00046 TABLE 7 Bacterial Diseases: Diphtheria Pertussis
Occult Bacteremia Urinary Tract Infection Gastroenteritis
Cellulitis Epiglottitis Tracheitis Adenoid Hypertrophy
Retropharyngeal Abcess Impetigo Ecthyma Pneumonia Endocarditis
Septic Arthritis Pneumococcal Peritonitis Bactermia Meningitis
Acute Purulent Meningitis Urethritis Cervicitis Proctitis
Pharyngitis Salpingitis Epididymitis Gonorrhea Syphilis Listeriosis
Anthrax Nocardiosis Salmonella Typhoid Fever Dysentery
Conjunctivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic
Plague Tetanus Necrotizing Enteritis Actinomycosis Mixed Anaerobic
Infections Syphilis Relapsing Fever Leptospirosis Lyme Disease Rat
Bite Fever Tuberculosis Lymphadenitis Leprosy Chlamydia Chlamydial
Pneumonia Trachoma Inclusion Conjunctivitis Systemic Fungal
Diseases: Histoplamosis Coccidiodomycosis Blastomycosis
Sporotrichosis Cryptococcsis Systemic Candidiasis Aspergillosis
Mucormycosis Mycetoma Chromomycosis Rickettsial Diseases: Typhus
Rocky Mountain Spotted Fever Ehrlichiosis Eastern Tick-Borne
Rickettsioses Rickettsialpox Q Fever Bartonellosis Parasitic
Diseases: Malaria Babesiosis African Sleeping Sickness Chagas'
Disease Leishmaniasis Dum-Dum Fever Toxoplasmosis
Meningoencephalitis Keratitis Entamebiasis Giardiasis
Cryptosporidiasis Isosporiasis Cyclosporiasis Microsporidiosis
Ascariasis Whipworm Infection Hookworm Infection Threadworm
Infection Ocular Larva Migrans Trichinosis Guinea Worm Disease
Lymphatic Filariasis Loiasis River Blindness Canine Heartworm
Infection Schistosomiasis Swimmer's Itch Oriental Lung Fluke
Oriental Liver Fluke Fascioliasis Fasciolopsiasis Opisthorchiasis
Tapeworm Infections Hydatid Disease Alveolar Hydatid Disease Viral
Diseases: Measles Subacute sclerosing panencephalitis Common Cold
Mumps Rubella Roseola Fifth Disease Chickenpox Respiratory
syncytial virus infection Croup Bronchiolitis Infectious
Mononucleosis Poliomyelitis Herpangina Hand-Foot-and-Mouth Disease
Bornholm Disease Genital Herpes Genital Warts Aseptic Meningitis
Myocarditis Pericarditis Gastroenteritis Acquired Immunodeficiency
Syndrome (AIDS) Human Immunodeficiency Virus (HIV) Reye's Syndrome
Kawasaki Syndrome Influenza Bronchitis Viral "Walking" Pneumonia
Acute Febrile Respiratory Disease Acute pharyngoconjunctival fever
Epidemic keratoconjunctivitis Herpes Simplex Virus 1 (HSV-1) Herpes
Simplex Virus 2 (HSV-2) Shingles Cytomegalic Inclusion Disease
Rabies Progressive Multifocal Leukoencephalopathy Kuru Fatal
Familial Insomnia Creutzfeldt-Jakob Disease
Gerstmann-Straussler-Scheinker Disease Tropical Spastic Paraparesis
Western Equine Encephalitis California Encephalitis St. Louis
Encephalitis Yellow Fever Dengue Lymphocytic choriomeningitis Lassa
Fever Hemorrhagic Fever Hantvirus Pulmonary Syndrome Marburg Virus
Infections Ebola Virus Infections Smallpox
4.8.7 Multi-Drug Therapy of Infectious Diseases
Methods for treating an infectious disease are disclosed including
administering to a patient in need thereof an Exemplary Conjugate
and another therapeutic agent that is an anti-infectious disease
agent. In one embodiment, the anti-infectious disease agent is, but
not limited to, agents listed in Table 8.
TABLE-US-00047 TABLE 8 .beta.-Lactam Antibiotics: Penicillin G
Penicillin V Cloxacilliin Dicloxacillin Methicillin Nafcillin
Oxacillin Ampicillin Amoxicillin Bacampicillin Azlocillin
Carbenicillin Mezlocillin Piperacillin Ticarcillin Aminoglycosides:
Amikacin Gentamicin Kanamycin Neomycin Netilmicin Streptomycin
Tobramycin Macrolides: Azithromycin Clarithromycin Erythromycin
Lincomycin Clindamycin Tetracyclines: Demeclocycline Doxycycline
Minocycline Oxytetracycline Tetracycline Quinolones: Cinoxacin
Nalidixic Acid Fluoroquinolones: Ciprofloxacin Enoxacin
Grepafloxacin Levofloxacin Lomefloxacin Norfloxacin Ofloxacin
Sparfloxacin Trovafloxicin Polypeptides: Bacitracin Colistin
Polymyxin B Sulfonamides: Sulfisoxazole Sulfamethoxazole
Sulfadiazine Sulfamethizole Sulfacetamide Miscellaneous
Antibacterial Agents: Trimethoprim Sulfamethazole Chloramphenicol
Vancomycin Metronidazole Quinupristin Dalfopristin Rifampin
Spectinomycin Nitrofurantoin Antiviral Agents: General Antiviral
Agents: Idoxuradine Vidarabine Trifluridine Acyclovir Famcicyclovir
Pencicyclovir Valacyclovir Gancicyclovir Foscarnet Ribavirin
Amantadine Rimantadine Cidofovir Antisense Oligonucleotides
Immunoglobulins Inteferons Drugs for HIV infection: Tenofovir
Emtricitabine Zidovudine Didanosine Zalcitabine Stavudine
Lamivudine Nevirapine Delavirdine Saquinavir Ritonavir Indinavir
Nelfinavir
5. EXAMPLES
Example 1
Preparation of Compound AB
##STR00052##
Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No.
6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M)
and to this solution was added a diethylamine (60 mL). The reaction
was monitored by HPLC and found to be complete in 2 h. The reaction
mixture was concentrated and the resulting residue was precipitated
using ethyl acetate (ca. 100 mL) under sonication over for 10 min.
Ether (200 mL) was added and the precipitate was further sonicated
for 5 min. The solution was allowed to stand for 30 min. without
stirring and was then filtered and dried under high vacuum to
provide Val-cit-PAB-OH, which was used in the next step without
further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g,
21 mmol) was diluted with DMF (110 mL) and the resulting solution
was treated with MC-OSu (Winner et al., (1993) Bioconjugate Chem.
4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to
HPLC after 2 h. The reaction mixture was concentrated and the
resulting oil was precipitated using ethyl acetate (50 mL). After
sonicating for 15 min, ether (400 mL) was added and the mixture was
sonicated further until all large particles were broken up. The
solution was then filtered and the solid dried to provide an
off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9
[M-H]
Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No.
6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M)
and to this solution was added a diethylamine (60 mL). The reaction
was monitored by HPLC and found to be complete in 2 h. The reaction
mixture was concentrated and the resulting residue was precipitated
using ethyl acetate (ca. 100 mL) under sonication over for 10 min.
Ether (200 mL) was added and the precipitate was further sonicated
for 5 min. The solution was allowed to stand for 30 min. without
stirring and was then filtered and dried under high vacuum to
provide Val-cit-PAB-OH, which was used in the next step without
further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g,
21 mmol) was diluted with DMF (110 mL) and the resulting solution
was treated with MC-OSu (Willner et al., (1993) Bioconjugate Chem.
4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to
HPLC after 2 h. The reaction mixture was concentrated and the
resulting oil was precipitated using ethyl acetate (50 mL). After
sonicating for 15 min, ether (400 mL) was added and the mixture was
sonicated further until all large particles were broken up. The
solution was then filtered and the solid dried to provide an
off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9
[M-H].
The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted
with DMF (120 mL, 0.12 M) and to the resulting solution was added
bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA
(3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h
according to HPLC. The reaction mixture was concentrated to provide
an oil that was precipitated with EtOAc, and then triturated with
EtOAc (ca. 25 mL). The solute was further precipitated with ether
(ca. 200 mL) and triturated for 15 min. The solid was filtered and
dried under high vacuum to provide Compound AB which was 93% pure
according to HPLC and used in the next step without further
purification. Yield: 9.7 g (94%).
Example 2
Preparation of Compound 1
##STR00053##
Phenylalanine t-butyl ester HCl salt (868 mg, 3 mmol),
N-Boc-Dolaproine (668 mg, 1 eq.), DEPC (820 .mu.L, 1.5 eq.), and
DIEA (1.2 mL) were diluted with dichloromethane (3 mL). After 2
hours (h) at room temperature (about 28 degrees Celsius), the
reaction mixture was diluted with dichloromethane (20 mL), washed
successively with saturated aqueous (aq.) NaHCO.sub.3 (2.times.10
mL), saturated aq. NaCl (2.times.10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography in ethyl
acetate. The relevant fractions were combined and concentrated to
provide the dipeptide as a white solid: 684 mg (46%). ES-MS m/z
491.3 [M+H].sup.+.
For selective Boc cleavage in the presence of t-butyl ester, the
above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2
mL). 4M HCl/dioxane (960 .mu.L, 3 eq.) was added, and the reaction
mixture was stirred overnight at room temperature. Almost complete
Boc deprotection was observed by RP-HPLC with minimal amount of
t-butyl ester cleavage. The mixture was cooled down on an ice bath,
and triethylamine (500 .mu.L) was added. After 10 min., the mixture
was removed from the cooling bath, diluted with dichloromethane (20
mL), washed successively with saturated aq. NaHCO.sub.3 (2.times.10
mL), saturated aq. NaCl (2.times.10 mL). The organic layer was
concentrated to give a yellow foam: 287 mg (57%). The intermediate
was used without further purification.
The tripeptide Fmoc-Meval-val-dil-O-t-Bu (prepared as described in
WO 02/088172, entitled "Pentapeptide Compounds and Uses Related
Thereto"; 0.73 mmol) was treated with TFA (3 mL), dichloromethane
(3 mL) for 2 h at room temperature. The mixture was concentrated to
dryness, the residue was co-evaporated with toluene (3.times.20
mL), and dried in vacuum overnight. The residue was diluted with
dichloromethane (5 mL) and added to the deprotected dipeptide (287
mg, 0.73 mmol), followed by DIEA (550 .mu.L, 4 eq.), DEPC (201
.mu.L, 1.1 eq.). After 2 h at room temperature the reaction mixture
was diluted with ethyl acetate (50 mL), washed successively with
10% aq. citric acid (2.times.20 mL), saturated aq. NaHCO.sub.3
(2.times.10 mL), saturated aq. NaCl (10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography in ethyl
acetate. The relevant fractions were combined and concentrated to
provide Fmoc-Meval-val-dil-dap-phe-O-t-Bu as a white solid: 533 mg
(71%). R.sub.f 0.4 (EtOAc). ES-MS m/z 1010.6 [M+H].sup.+.
The product (200 mg, 0.2 mmol) was diluted with dichloromethane (3
mL), diethylamine (1 mL). The reaction mixture was stirred
overnight at room temperature. Solvents were removed to provide an
oil that was purified by flash silica gel chromatography in a step
gradient 0-10% MeOH in dichloromethane to provide Compound 1 as a
white solid: 137 mg (87%). R.sub.f 0.3 (10% MeOH/CH.sub.2Cl.sub.2).
ES-MS m/z 788.6 [M+H].sup.+.
Example 3
Preparation of Compound 2
##STR00054##
Compound 2 was prepared from compound 1 (30 mg, 0.038 mmol) by
treatment with 4M HCl/dioxane (4 ml) for 7 h at room temperature.
The solvent was removed, and the residue was dried in a vacuum
overnight to give provide Compound 2 as a hydroscopic white solid:
35 mg (120% calculated for HCl salt). ES-MS m/z 732.56
[M+H].sup.+.
Example 4
Preparation of Compound 3
##STR00055##
Fmoc-Meval-val-dil-dap-phe-O-t-Bu (Example 2, 50 mg) was treated
with 4M HCl/dioxane (4 ml) for 16 h at room temperature. The
solvent was removed, and the residue was dried in vacuum overnight
to give 50 mg of a hydroscopic white solid intermediate
The white solid intermediate (20 mg, 0.02 mmol) was diluted with
dichloromethane (1 mL); DEPC (5 .mu.L, 0.03 mmol, 1.5 eq.) was
added followed by DIEA (11 .mu.L, 0.06 mmol, 3 eq.), and
t-butylamine (3.2 .mu.L, 0.03 mmol, 1.5 eq.). After 2 h at room
temperature, the reaction was found to be uncompleted by RP-HPLC.
More DEPC (10 .mu.l) and t-butylamine (5 .mu.L) were added and the
reaction was stirred for additional 4 h. Reaction mixture was
diluted with dichloromethane (15 mL), washed successively with
water (5 mL), 0.1 M aq. HCl (10 mL), saturated aq. NaCl (10 mL).
The organic layer was separated and concentrated. The resulting
residue was diluted with dichloromethane and purified via flash
chromatography in a step gradient 0-5% MeOH in dichloromethane. The
relevant fractions were combined and concentrated to provide the
Fmoc protected intermediate as a white solid: 7.3 mg (36%). R.sub.f
0.75 (10% MeOH/CH.sub.2Cl.sub.2).
Fmoc protected intermediate was diluted with dichloromethane (0.5
mL) and treated with diethylamine (0.5 mL) for 3 h at room
temperature. The reaction mixture was concentrated to dryness. The
product was isolated by flash silica gel chromatography in a step
gradient 0-10% MeOH in dichloromethane to provide Compound 3 as a
white solid: 4 mg (70%). R.sub.f 0.2 (10% MeOH/CH.sub.2Cl.sub.2).
ES-MS m/z 787 [M+H].sup.+, 809 [M+Na].sup.+.
Example 5
Preparation of Compound 4
##STR00056##
Boc-L-Phenylalanine (265 mg, 1 mmol, 1 eq.) and triethyleneglycol
monomethyl ether (164 .mu.L, 1 mmol, 1 eq.) were diluted with
dichloromethane (5 mL). Then, DCC (412 mg, 2 mmol, 2 eq.) was
added, followed by DMAP (10 mg). The reaction mixture was stirred
overnight at room temperature. The precipitate was filtered off.
The solvent was removed in a vacuum, the residue was diluted with
ethyl acetate, and purified by silica gel flash chromatography in
ethyl acetate. The product containing fractions were pulled,
concentrated, and dried in vacuum to give a white solid: 377 mg
(91%). R.sub.f 0.5 (EtOAc). ES-MS m/z 434 [M+Na].sup.+.
Removal of Boc protecting group was performed by treatment of the
above material in dioxane (10 mL) with 4M HCl/dioxane (6 mL) for 6
h at room temperature. The solvent was removed in a vacuum, the
residue was dried in a vacuum to give a white solid.
The HCl salt of Phenylalanine-triethyleneglycol monomethyl ether
ester (236 mg, 0.458 mmol, 1 eq.) and N-Boc-Dolaproine (158 mg,
0.55 mmol, 1.2 eq.) were diluted with dichloromethane (3 mL). DEPC
(125 .mu.L, 1.5 eq.) and added to the mixture followed by DIEA (250
.mu.L, 3 eq.). After 2 h at room temperature the reaction mixture
was diluted with ethyl acetate (30 mL), washed successively with
saturated aq. NaHCO.sub.3 (2.times.10 mL), 10% aq. citric acid
(2.times.10 mL), saturated aq. NaCl (10 mL). The organic layer was
separated and concentrated. The resulting residue was re-suspended
in ethyl acetate and was purified via flash chromatography on
silica gel in ethyl acetate. The relevant fractions were combined
and concentrated to provide a white foam intermediate: 131 mg
(50%). R.sub.f 0.25 (EtOAc). ES-MS m/z 581.3 [M+H].sup.+.
Boc deprotection was done in dichloromethane (2 mL), TFA (0.5 mL)
at room temperature for 2 h. Solvent was removed in vacuum, and the
residue was co-evaporated with toluene (3.times.25 mL), then dried
in vacuum to give 138 mg of dipeptide TFA salt.
Fmoc-Meval-val-dil-OH (Example 2, 147 mg, 0.23 mmol, 1 eq.), and
dipeptide TFA salt (138 mg) were diluted with dichloromethane (2
mL). To the mixture DEPC (63 .mu.L, 1.5 eq.) was added, followed by
DIEA (160 .mu.L, 4 eq.). After 2 h at room temperature the reaction
mixture was diluted with dichloromethane (30 mL), washed
successively with 10% aq. citric acid (2.times.20 mL), saturated
aq. NaCl (20 mL). The organic layer was separated and concentrated.
The resulting residue was re-suspended in dichloromethane and was
purified via flash chromatography on silica gel in a step gradient
0-5% MeOH in dichloromethane. The relevant fractions were combined
and concentrated to provide white foam: 205 mg (81%). R.sub.f 0.4
(10% MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 1100.6 [M+H].sup.+, 1122.4
[M+Na].sup.+.
Fmoc protecting group was removed by treatment with diethylamine (2
mL) in dichloromethane (6 mL). After 6 h at room temperature
solvent was removed in vacuum, product was isolated by flash
chromatography on silica gel in a step gradient 0-10% MeOH in
dichloromethane. The relevant fractions were combined and
concentrated. After evaporation from dichloromethane/hexane, 1:1,
Compound 4 was obtained as a white foam: 133 mg (80%). R.sub.f 0.15
(10% MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 878.6 [M+H].sup.+.
Example 6
Preparation of Compound 5
##STR00057##
Fmoc-Meval-val-dil-OH (Example 2, 0.50 g, 0.78 mmol) and
dap-phe-OMe.HCl (0.3 g, 0.78 mmol, prepared according to Pettit, G.
R., et al. Anti-Cancer Drug Design 1998, 13, 243-277) were
dissolved in CH.sub.2Cl.sub.2 (10 mL) followed by the addition of
diisopropylethylamine (0.30 mL, 1.71 mmol, 2.2 eq.). DEPC (0.20 mL,
1.17, 1.5 eq.) was added and the contents stood over Ar. Reaction
was complete according to HPLC in 1 h. The mixture was concentrated
to an oil and purified by SiO.sub.2 chromatography (300.times.25 mm
column) and eluting with 100% EtOAc. The product was isolated as a
white foamy solid. Yield: 0.65 g (87%). ES-MS m/z 968.35
[M+H].sup.+, 991.34 [M+Na].sup.+; UV .lamda..sub.max 215, 265
nm.
The Fmoc-protected peptide (0.14 g, 0.14 mmol) in methylene
chloride (5 mL) was treated with diethylamine (2 mL) and the
contents stood at room temperature for 2 h. The reaction, complete
by HPLC, was concentrated to an oil, taken up in 2 mL of DMSO and
injected into a preparative-HPLC (C.sub.12-RP column, 5.mu., 100
.ANG., linear gradient of MeCN in water (containing 0.1% TFA) 10 to
100% in 40 min followed by 20 min at 100%, at a flow rate of 25
mL/min). Fractions containing the product were evaporated to afford
a white powder for the trifluoroacetate salt. Yield: 0.126 g (98%).
R.sub.f 0.28 (100% EtOAc); ES-MS m/z 746.59 [M+H].sup.+, 768.51
[M+Na].sup.+; UV .lamda..sub.max 215 nm.
Example 7
Preparation of Compound 6
##STR00058##
The trifluoroacetate salt of Compound 5 (0.11 g, 0.13 mmol),
Compound AB (0.103 g, 0.14 mmol, 1.1 eq.) and HOBt (3.4 mg, 26
.mu.mol, 0.2 eq.) were suspended in DMF/pyridine (2 mL/0.5 mL,
respectively). Diisopropylethylamine (22.5 .mu.L, 0.13 mmol, 1.0
eq.) was added and the yellow solution stirred while under argon.
After 3 h, an additional 1.0 eq. of DIEA was added. 24 hours later,
0.5 eq. of the activated linker was included in the reaction
mixture. After 40 h total, the reaction was complete. The contents
were evaporated, taken up in DMSO and injected into a prep-HPLC
(C.sub.12-RP column, 5.mu., 100 .ANG., linear gradient of MeCN in
water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min
at 100%, at a flow rate of 50 mL/min). The desired fractions were
evaporated to give the product as a yellow oil. Methylene chloride
(ca. 2 mL) and excess ether were added to provide Compound 6 as a
white precipitate that was filtered and dried. Yield: 90 mg (52%).
ES-MS m/z 1344.32 [M+H].sup.+, 1366.29 [M+Na].sup.+; UV
.lamda..sub.max 215, 248 nm.
Example 8
Preparation of Compound 7
##STR00059##
Compound 4 (133 mg, 0.15 mmol, 1 eq.), Compound AB, (123 mg, 0.167
mmol, 1.1 eq.), and HOBt (4 mg, 0.2 eq.) were diluted with DMF (1.5
mL). After 2 min, pyridine (5 mL) was added and the reaction was
monitored using RP-HPLC. The reaction was shown to be complete
within 18 h. The reaction mixture was diluted with dichloromethane
(20 mL), washed successively with 10% aq. citric acid (2.times.10
mL), water (10 mL), saturated aq. NaCl (10 mL). The organic layer
was separated and concentrated. The resulting residue was
re-suspended in dichloromethane and was purified via flash
chromatography on silica gel in a step gradient 0-10% MeOH in
dichloromethane. The relevant fractions were combined and
concentrated to provide Compound 7 as a white foam: 46 mg (21%).
R.sub.f 0.15 (10% MeOH/CH.sub.2Cl.sub.2). ES-MS m/z 1476.94
[M+H].sup.+.
Example 9
Preparation of MC-Val-Cit-PAB-MMAF t-butyl ester 8
##STR00060##
Compound 1 (83 mg, 0.11 mmol), Compound AB (85 mg, 0.12 mmol, 1.1
eq.), and HOBt (2.8 mg, 21 .mu.mmol, 0.2 eq.) were taken up in dry
DMF (1.5 mL) and pyridine (0.3 mL) while under argon. After 30 h,
the reaction was found to be essentially complete by HPLC. The
mixture was evaporated, taken up in a minimal amount of DMSO and
purified by prep-HPLC (C.sub.12-RP column, 5.mu., 100 .ANG., linear
gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40
min followed by 20 min at 100%, at a flow rate of 25 mL/min) to
provide Compound 8 as a white solid. Yield: 103 mg (71%). ES-MS m/z
1387.06 [M+H].sup.+, 1409.04 [M+Na].sup.+; UV .lamda..sub.max 205,
248 nm.
Example 10
Preparation of MC-val-cit-PAB-MMAF 9
##STR00061##
Compound 8 (45 mg, 32 mmol) was suspended in methylene chloride (6
mL) followed by the addition of TFA (3 mL). The resulting solution
stood for 2 h. The reaction mixture was concentrated in vacuo and
purified by prep-HPLC (C.sub.12-RP column, 5.mu., 100 .ANG., linear
gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40
min followed by 20 min at 100%, at a flow rate of 25 mL/min). The
desired fractions were concentrated to provide
maleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF
(MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%).
ES-MS m/z 1330.29 [M+H].sup.+, 1352.24 [M+Na].sup.+; UV
.lamda..sub.max 205, 248 nm.
Example 11
Preparation of MC-val-cit-PAB-MMAF tert-butyl amide 10
##STR00062##
Compound 3 (217 mg, 0.276 mmol, 1.0 eq.), Compound AB (204 mg,
0.276 mmol, 1.0 eq.), and HOBt (11 mg, 0.0828 mmol, 0.3 eq.) were
diluted with pyridine/DMF (6 mL). To this mixture was added DIEA
(0.048 mL), and the mixture was stirred ca. 16 hr. Volatile
organics were evaporated in vacuo. The crude residue was purified
by Chromatotron.RTM. (radial thin-layer chromatography) with a step
gradient (0-5-10% methanol in DCM) to provide MC-val-cit-PAB-MMAF
tert-butyl amide 10. Yield: 172 mg (45%); ES-MS m/z 1386.33
[M+H].sup.+, 1408.36 [M+Na].sup.+; UV .lamda..sub.max 215, 248
nm.
Example 12
Preparation of AC10-MC-MMAE by Conjugation of AC10 and MC-MMAE
AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride
at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT).
After incubation at 37.degree. C. for about 30 minutes, the buffer
is exchanged by elution over Sephadex G25 resin and eluted with PBS
with 1 mM DTPA. The thiol/Ab value is checked by determining the
reduced antibody concentration from the absorbance at 280 nm of the
solution and the thiol concentration by reaction with DTNB
(Aldrich, Milwaukee, Wis.) and determination of the absorbance at
412 nm. The reduced antibody dissolved in PBS is chilled on
ice.
The drug linker reagent, maleimidocaproyl-monomethyl auristatin E,
i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and
water at known concentration, and added to the chilled reduced
antibody AC10 in PBS. After about one hour, an excess of maleimide
is added to quench the reaction and cap any unreacted antibody
thiol groups. The reaction mixture is concentrated by centrifugal
ultrafiltration and AC10-MC-MMAE is purified and desalted by
elution through G25 resin in PBS, filtered through 0.2 .mu.m
filters under sterile conditions, and frozen for storage.
Example 13
Preparation of AC10-MC-MMAF by Conjugation of AC10 and MC-MMAF
AC10-MC-MMAF was prepared by conjugation of AC10 and MC-MMAF
following the procedure of Example 12.
Example 14
Preparation of AC10-MC-val-cit-PAB-MMAE by Conjugation of AC10 and
MC-val-cit-PAB-MMAE
AC10-MC-val-cit-PAB-MMAE was prepared by conjugation of AC10 and
MC-val-cit-PAB-MMAE following the procedure of Example 12.
Example 15
Preparation of AC10-MC-val-cit-PAB-MMAF by Conjugation of AC10 and
MC-val-cit-PAB-MMAF (9)
AC10-MC-val-cit-PAB-MMAF was prepared by conjugation of AC10 and
MC-val-cit-PAB-MMAF (9) following the procedure of Example 12.
Example 16
Determination of Cytotoxicity of Selected Compounds
Cytotoxic activity of MMAF and Compounds 1-5 was evaluated on the
Lewis Y positive cell lines OVCAR-3, H3396 breast carcinoma, L2987
lung carcinoma and LS174t colon carcinoma Lewis Y positive cell
lines can be assayed for cytotoxicity. To evaluate the cytotoxicity
of Compounds 1-5, cells can be seeded at approximately 5-10,000 per
well in 150 .mu.I of culture medium then treated with graded doses
of Compounds 1-5 in quadruplicates at the initiation of assay.
Cytotoxicity assays are usually carried out for 96 hours after
addition of test compounds. Fifty .mu.l of resazurin dye may be
added to each well during the last 4 to 6 hours of the incubation
to assess viable cells at the end of culture. Dye reduction can be
determined by fluorescence spectrometry using the excitation and
emission wavelengths of 535 nm and 590 nm, respectively. For
analysis, the extent of resazurin reduction by the treated cells
can be compared to that of the untreated control cells.
For 1 h exposure assays cells can be pulsed with the drug for 1 h
and then washed; the cytotoxic effect can be determined after 96 h
of incubation.
Example 17
In Vitro Cytotoxicity Cata for Selected Compounds
Table 10 shows cytotoxic effect of cAC10 Conjugates of Compounds
7-10, assayed as described in General Procedure I on a CD30+ cell
line Karpas 299. Data of two separate experiments are presented.
The cAC10 conjugates of Compounds 7 and 9 were found to be slightly
more active than cAC10-val-cit-MMAE.
TABLE-US-00048 TABLE 10 Conjugate IC.sub.50 (ng/mL)
cAC10-val-cit-MMAE 6 cAC10-7 1.0 cAC10-8 15 cAC10-9 0.5 cAC10-10
20
In other experiments, BR96-val-cit-MMAF was at least 250 fold more
potent than the free MMAF.
General Procedure 1--Cytotoxicity Determination.
To evaluate the cytotoxicity of Exemplary Conjugates 7-10, cells
were seeded at approximately 5-10,000 per well in 150 .mu.l of
culture medium then treated with graded doses of Exemplary
Conjugates 7-10 in quadruplicates at the initiation of assay.
Cytotoxicity assays were carried out for 96 hours after addition of
test compounds. Fifty .mu.l of the resazurin dye was added to each
well during the last 4 to 6 hours of the incubation to assess
viable cells at the end of culture. Dye reduction was determined by
fluorescence spectrometry using the excitation and emission
wavelengths of 535 nm and 590 nm, respectively. For analysis, the
extent of resazurin reduction by the treated cells was compared to
that of the untreated control cells.
Example 18
In Vitro Cell Proliferation Assay
Efficacy of ADC can be measured by a cell proliferation assay
employing the following protocol (Promega Corp. Technical Bulletin
TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488):
1. An aliquot of 100 .mu.l of cell culture containing about
10.sup.4 cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was
deposited in each well of a 96-well, opaque-walled plate.
2. Control wells were prepared containing medium and without
cells.
3. ADC was added to the experimental wells and incubated for 3-5
days.
4. The plates were equilibrated to room temperature for
approximately 30 minutes.
5. A volume of CellTiter-Glo Reagent equal to the volume of cell
culture medium present in each well was added.
6. The contents were mixed for 2 minutes on an orbital shaker to
induce cell lysis.
7. The plate was incubated at room temperature for 10 minutes to
stabilize the luminescence signal.
8. Luminescence was recorded and reported in graphs as RLU=relative
luminescence units.
Example 19
Plasma Clearance in Rat
Plasma clearance pharmacokinetics of antibody drug conjugates and
total antibody was studied in Sprague-Dawley rats (Charles River
Laboratories, 250-275 gms each). Animals were dosed by bolus tail
vein injection (IV Push). Approximately 300 whole blood was
collected through jugular cannula, or by tail stick, into
lithium/heparin anticoagulant vessels at each timepoint: 0
(predose), 10, and 30 minutes; 1, 2, 4, 8, 24 and 36 hours; and 2,
3, 4, 7, 14, 21, 28 days post dose. Total antibody was measured by
ELISA-ECD/GxhuFc-HRP. Antibody drug conjugate was measured by
ELISA-MMAE/MMAF/ECD-Bio/SA-HRP.
Example 20
Plasma Clearance in Monkey
Plasma clearance pharmacokinetics of antibody drug conjugates and
total antibody can be studied in cynomolgus monkeys. FIG. 12 shows
a two-stage plasma concentration clearance study after
administration of H-MC-vc-MMAE to Cynomolgus monkeys at different
doses: 0.5, 1.5, 2.5, and 3.0 mg/kg, administered at day 1 and day
21. Concentrations of total antibody and ADC were measured over
time. (H=Trastuzumab).
Example 21
Tumor Volume In Vivo Efficacy in Transgenic Explant Mice
Animals suitable for transgenic experiments can be obtained from
standard commercial sources such as Taconic (Germantown, N.Y.).
Many strains are suitable, but FVB female mice are preferred
because of their higher susceptibility to tumor formation. FVB
males can be used for mating and vasectomized CD.1 studs can be
used to stimulate pseudopregnancy. Vasectomized mice can be
obtained from any commercial supplier. Founders can be bred with
either FVB mice or with 129/BL6.times.FVB p53 heterozygous mice.
The mice with heterozygosity at p53 allele can be used to
potentially increase tumor formation. Some F1 tumors are of mixed
strain. Founder tumors can be FVB only.
Animals having tumors (allograft propagated from Fo5 mmtv
transgenic mice) can be treated with a single or multiple dose by
IV injection of ADC. Tumor volume can be assessed at various time
points after injection.
Example 22
Synthesis of MC-MMAF Via t-butyl ester
MeVal-Val-Dil-Dap-Phe-OtBu (compound I, 128.6 mg, 0.163 mmol) was
suspended in CH.sub.2Cl.sub.2 (0.500 mL). 6-Maleimidocaproic acid
(68.9 mg, 0.326 mmol) and 1,3-diisopropylcarbodiimide (0.0505 mL,
0.326 mmol) were added followed by pyridine (0.500 mL). Reaction
mixture was allowed to stir for 1.0 hr. HPLC analysis indicated
complete consumption of starting compound 1. Volatile organics were
evaporated under reduced pressure. Product was isolated via flash
column chromatography, using a step gradient from 0 to 5% Methanol
in CH.sub.2Cl.sub.2. A total of 96 mg of pure
MC-MeVal-Val-Dil-Dap-Phe-OtBu (12) (60% yield) was recovered. ES-MS
m/z 981.26 [M+H].sup.+; 1003.47 [M+Na].sup.+; 979.65 [M-H].sup.-.
See FIG. 37.
MC-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 12, 74 mg, 0.0754 mmol) was
suspended in CH.sub.2Cl.sub.2 (2.0 mL) and TFA (1 mL) at room
temperature. After 2.5 hr, HPLC analysis indicated complete
consumption of starting material. Volatile organics were evaporated
under reduced pressure, and the product was isolated via
preparatory RP-HPLC, using a Phenomenex C.sub.12 Synergi Max-RP 80
.ANG. Column (250.times.21.20 mm). Eluent: linear gradient 10% to
90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90%
MeCN/0.05% TFA (aq) for an additional 20 minutes. ES-MS m/z 925.33
[M+H].sup.+; 947.30 [M+Na].sup.+; 923.45 [M-H].sup.-.
Example 23a
Synthesis of MC-MMAF (11) Via dimethoxybenzyl ester
Preparation of Fmoc-L-Phenylalanine-2,4-dimethoxybenzyl ester
(Fmoc-Phe-ODMB) See FIG. 38.
A 3-neck, 5-L round-bottom flask was charged with
Fmoc-L-Phenylalanine (200 g, 516 mmol Bachem), 2,4-dimethoxybenzyl
alcohol (95.4 g, 567 mmol, Aldrich), and CH.sub.2Cl.sub.2 (2.0 L).
N,N-dimethylformamide t-butyl acetal (155 mL, 586 mmol, Fluka) was
added to the resulting suspension over 20 min under N.sub.2, which
resulted in a clear solution. The reaction was then stirred at room
temperature overnight, after which time TLC analysis (0.42,
Heptane/EtOAc=2:1) indicated that the reaction was complete. The
reaction mixture was concentrated under reduced pressure to give a
light yellow oil, which was redissolved in CH.sub.2Cl.sub.2 (200
mL) and purified through a short plug of silica gel (25 cm.times.25
cm, CH.sub.2Cl.sub.2) to give a colorless foam (250 g). MeCN (1 L)
was added into the resulting foam, which totally dissolved the
batch. It was then concentrated to dryness and redissolved in MeCN
(1 L) and the resulting suspension was stirred for 1 h, filtered
and the filter cake was rinsed with MeCN (2.times.200 mL) to give
Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester as a white solid
(113.58 g, 41%, 95.5% AUC by HPLC analysis). Data: HPLC.
Preparation L-Phenylalanine-2,4-dimethoxybenzyl ester
(Phe-ODMB)
A 500-mL round-bottom flask was charged with
Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester (26.00 g, 48.3
mmol), CH.sub.2Cl.sub.2 (150 mL) and diethylamine (75 mL, Acros).
Mixture was stirred at room temperature and the completion
monitored by HPLC. After 4 h, the mixture was concentrated (bath
temp<30.degree. C.). The residue was resuspended in
CH.sub.2Cl.sub.2 (200 mL) and concentrated. This was repeated once.
To the residue was added MeOH (20 mL), which caused the formation
of a gel. This residue was diluted with CH.sub.2Cl.sub.2 (200 mL),
concentrated and the cloudy oil left under vacuum overnight. The
residue was suspended in CH.sub.2Cl.sub.2 (100 mL), then toluene
(120 mL) was added. The mixture was concentrated and the residue
left under vacuum overnight.
Data: HPLC, 1H NMR.
Preparation of Fmoc-Dolaproine (Fmoc-Dap)
Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HCl in
1,4-dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5
hours, TLC analysis indicated the reaction was complete (10%
MeOH/CH.sub.2Cl.sub.2) and the mixture was concentrated to
near-dryness. Additional 1,4-dioxane was charged (50 mL) and the
mixture was concentrated to dryness and dried under vacuum
overnight. The resulting white solid was dissolved in H.sub.2O (400
mL) and transferred to a 3-L, three-neck, round-bottom flask with a
mechanical stirrer and temperature probe. N,N-diisopropylethylamine
(214.3 mL, 1.23 mol, Acros) was added over one minute, causing an
exotherm from 20.5 to 28.2.degree. C. (internal). The mixture was
cooled in an ice bath and 1,4-dioxane was added (400 mL). A
solution of Fmoc-OSu (89.90 g, 0.267 mol, Advanced ChemTech) in
1,4-dioxane (400 mL) was added from an addition funnel over 15
minutes, maintaining the reaction temperature below 9.degree. C.
The mixture was allowed to warm to room temperature and stir for 19
hours, after which the mixture was concentrated by rotary
evaporation to an aqueous slurry (390 g). The suspension was
diluted with H.sub.2O (750 mL) and Et.sub.2O (750 mL), causing a
copious white precipitate to form. The layers were separated,
keeping the solids with the organic layer. The aqueous layer was
acidified using conc. HCl (30 mL) and extracted with EtOAc
(3.times.500 mL). The combined extracts were dried over MgSO.sub.4,
filtered and concentrated to give 59.25 g of a yellow oil A. The
Et.sub.2O extract was extracted once with sat. NaHCO.sub.3 (200
mL), keeping the solids with the aqueous layer. The aqueous
suspension was acidified using conc. HCl (50 mL) and extracted with
Et.sub.2O (50 mL) keeping the solids with the organic layer. The
organic layer was filtered and concentrated to give 32.33 g of a
yellow oil B. The two oils (A and B) were combined and purified by
flash chromatography on silica gel eluting with CH.sub.2Cl.sub.2
(3.5 L), then 3% MeOH/CH.sub.2Cl.sub.2 (9 L) to give 68.23 g of
Fmoc-dolaproine as a white foam (81%, 97.5% purity by HPLC
(AUC)).
Preparation of Fmoc-Dap-Phe-ODMB
Crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 mL,
Acros) for 5 minutes and Fmoc-Dap (19.80 g, 48.3 mmol) was added.
The mixture was cooled in an ice bath and TBTU (17.08 g, 53.20
mmol, Matrix Innovations) was added. N,N-diisopropylethylamine
(25.3 mL, 145.0 mmol, Acros) was added via syringe over 3 min.
After 1 h, the ice bath was removed and the mixture was allowed to
warm over 30 min. The mixture was poured into water (1 L) and
extracted with ethyl acetate (300 mL). After separation, the
aqueous layer was re-extracted with ethyl acetate (2.times.150 mL).
The combined organic layers were washed with brine (150 mL), dried
(MgSO4) and filtered (filter paper) to remove the insolubles
(inorganics and some dibenzofulvene). After concentration, the
residue (41 g) was adsorbed on silica (41 g) and purified by
chromatography (22 cm.times.8 cm column; 65% Heptane/EtOAc (2.5 L);
33% Heptane/EtOAc (3.8 L), to give 29.4 g of product as a white
foam (86%, 92% purity by HPLC).
Data: HPLC, .sup.1H NMR, TLC (1:1 EtOAc/Heptane Rf=0.33, red in
vanillin stain).
Preparation of Dap-Phe-ODMB
A 1-L round bottom flask was charged with Fmoc-Dap-Phe-ODMB (27.66
g), CH.sub.2Cl.sub.2 (122 mL) and diethylamine (61 mL, Acros). The
solution was stirred at room temperature and the completion
monitored by HPLC. After 7 h, the mixture was concentrated (bath
temp.<30.degree. C.). The residue was suspended in
CH.sub.2Cl.sub.2 (300 mL) and concentrated. This was repeated
twice. To the residue was added MeOH (20 mL) and CH.sub.2Cl.sub.2
(300 mL), and the solution was concentrated. The residue was
suspended in CH.sub.2Cl.sub.2 (100 mL) and toluene (400 mL),
concentrated, and the residue left under vacuum overnight to give a
cream-like residue.
Data: HPLC, 1H NMR, MS.
Preparation of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB
Crude Dap-Phe-ODMB (39.1 mmol) was suspended in anhydrous DMF (135
mL, Acros) for 5 minutes and Fmoc-MeVal-Val-Dil-OH (24.94 g, 39.1
mmol, see Example 2 for preparation) was added. The mixture was
cooled in an ice bath and TBTU (13.81 g, 43.0 mmol, Matrix
Innovations) was added. N,N-Diisopropylethylamine (20.5 mL, 117.3
mmol, Acros) was added via syringe over 2 minutes. After 1 hour,
the ice bath was removed and the mixture was allowed to warm over
30 min. The mixture was poured into water (1.5 L) and diluted with
ethyl acetate (480 mL). After standing for 15 minutes, the layers
were separated and the aqueous layer was extracted with ethyl
acetate (300 mL). The combined organic layers were washed with
brine (200 mL), dried (MgSO.sub.4) and filtered (filter paper) to
remove insolubles (inorganics and some dibenzofulvene). After
concentration, the residue (49 g) was scraped from the flask and
adsorbed on silica (49 g) and purified by chromatography (15
cm.times.10 cm dia column; 2:1 EtOAc/Heptane (3 L), EtOAc (5 L);
250 mL fractions) to give 31.84 g of
Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (73%, 93% purity by
HPLC (AUC)).
Data: HPLC, TLC (2:1 EtOAc/heptane, Rf=0.21, red in vanillin
stain).
Preparation of MeVal-Val-Dil-Dap-Phe-ODMB
A 1-L, round-bottom flask was charged with
Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB (28.50 g), CH.sub.2Cl.sub.2 (80 mL)
and diethylamine (40 mL). Mixture was stirred at room temperature
overnight and then was concentrated under reduced pressure. The
residue was adsorbed on silica (30 g) and purified by flash
chromatography (15 cm.times.8 cm dia column; 2% MeOH/DCM (2 L), 3%
MeOH/DCM (1 L), 6% MeOH/DCM (4 L); 250 mL fractions) to give 15.88
g of MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (69%, 96% purity by
HPLC (AUC)).
Data: HPLC, TLC (6% MeOH/DCM, Rf=0.24, red in vanillin stain).
Preparation of MC-MeVal-Val-Dil-Dap-Phe-ODMB
A 50-mL, round-bottom flask was charged with
MeVal-Val-Dil-Dap-Phe-ODMB (750 mg, 0.85 mmol), anhydrous DMF (4
mL), maleimidocaproic acid (180 mg, 0.85 mmol), and TBTU (300 mg,
0.93 mmol, Matrix Innovations) at room temperature.
N,N-Diisopropylethylamine (450 .mu.L, 2.57 mmol) was added via
syringe. After 1.5 hours, the mixture was poured in water (50 mL)
and diluted with ethyl acetate (30 mL). NaCl was added to improve
the separation. After separation of the layers, the aqueous layer
was extracted with ethyl acetate (25 mL). The combined organic
layers were dried (MgSO4), filtered and concentrated. The resulting
oil (1 g) was purified by flash chromatography [100 mL silica; 25%
Heptane/EtOAc (100 mL), 10% Heptane/EtOAc (200 mL), EtOAc (1.5 L)]
to give MC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg,
57%, 94% purity by HPLC(AUC)).
Data: 1H NMR, HPLC.
Preparation of MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF) (11)
A 50-mL, round-bottom flask was charged with
MC-MeVal-Val-Dil-Dap-Phe-ODMB (Compound 13, 428 mg, 0.39 mmol) and
dissolved in 2.5% TFA/CH.sub.2Cl.sub.2 (20 mL). The solution turned
pink-purple over 2 min. The completion was monitored by HPLC and
TLC (6% MeOH/DCM, KMnO.sub.4 stain). After 40 min, three drops of
water were added and the cloudy pink-purple mixture was
concentrated to give 521 mg of a pink residue. Purification by
chromatography (15% IPA/DCM) gave 270 mg of MC-MMAF (73%, 92%
purity by HPLC) as a white solid.
Example 23b
Synthesis of Analog of mc-MMAF
MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 35 mg, 0.044 mmol) was
suspended in DMF (0.250 mL).
4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic acid (11 mg, 0.049
mmol) and HATU (17 mg, 0.044 mmol) were added followed by DIEA
(0.031 mL, 0.17 mmol) See FIG. 39. This reaction mixture was
allowed to stir for 2.0 hr. HPLC analysis indicated complete
consumption of starting compound 1.
Product was isolated via preparatory RP-HPLC, using a Phenomenex
C.sub.12 Synergi Max-RP 80 .ANG. Column (250.times.21.20 mm).
Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8
minutes, then isocratic 80% MeCN/0.05% TFA (aq) for an additional
12 minutes. A total of 20 mg of pure product (14) was isolated
(0.02 mmol, 46% yield). ES-MS m/z 987.85 [M+H].sup.+; 1019.41
[M+Na].sup.+; 985.54 [M-H].sup.-.
MB-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 14, 38 mg, 0.0385 mmol) was
suspended in CH.sub.2Cl.sub.2 (1 mL) and TFA (1 mL). Mixture was
stirred for 2.0 hr, and then volatile organics were evaporated
under reduced pressure. Product was purified by preparatory
RP-HPLC, using a Phenomenex C.sub.12 Synergi Max-RP 80 .ANG. Column
(250.times.21.20 mm). Eluent: linear gradient 10% to 80% MeCN/0.05%
TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05% TFA (aq) for
an additional 12 minutes. A total of 14.4 mg of MB-MMAF product was
isolated (0.015 mmol, 40% yield). ES-MS m/z 930.96 [M+H].sup.+
952.98 [M+Na].sup.+; 929.37 [M-H].sup.-.
Example 23c
Preparation of MC-MeVal-Cit-PAS-MMAF (16)
##STR00063##
To a room temperature suspension of Fmoc-MeVal-OH (3.03 g, 8.57
mmol) and N,N'-disuccimidyl carbonate (3.29 g, 12.86 mmol) in
CH.sub.2Cl.sub.2 (80 mL) was added DIEA (4.48 mL, 25.71 mmol). This
reaction mixture was allowed to stir for 3.0 hr, and then poured
into a separation funnel where the organic mixture was extracted
with 0.1 M HCl (aq). The crude organic residue was concentrated
under reduced pressure, and the product was isolated by flash
column chromatography on silica gel using a 20-100% ethyl
acetate/hexanes linear gradient. A total of 2.18 g of pure
Fmoc-MeVal-OSu (4.80 mmoles, 56% yield) was recovered.
To a room temperature suspension of Fmoc-MeVal-OSu (2.18 g, 4.84
mmol) in DME (13 mL) and THF (6.5 mL) was added a solution of
L-citrulline (0.85 g, 4.84 mmol) and NaHCO.sub.3 (0.41 g, 4.84
mmol) in H.sub.2O (13 mL). The suspension was allowed to stir at
room temperature for 16 hr, then it was extracted into
tert-BuOH/CHCl.sub.3/H.sub.2O, acidified to pH=2-3 with 1 M HCl.
The organic phase was separated, dried and concentrated under
reduced pressure. The residue was triturated with diethyl ether
resulting in 2.01 g of Fmoc-MeVal-Cit-COOH which was used without
further purification.
The crude Fmoc-MeVal-Cit-COOH was suspended in 2:1
CH.sub.2Cl.sub.2/MeOH (100 mL), and to it was added p-aminobenzyl
alcohol (0.97 g, 7.9 mmol) and EEDQ (1.95 g, 7.9 mmol). This
suspension was allowed to stir for 125 hr, then the volatile
organics were removed under reduced pressure, and the residue was
purified by flash column chromatography on silica gel using a 10%
MeOH/CH.sub.2Cl.sub.2. Pure Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896
mmol, 18.5% yield) was recovered. ES-MS m/z 616.48 [M+H].sup.+.
To a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol) in
CH.sub.2Cl.sub.2 (40 mL) was added STRATOSPHERES.TM.
(piperizine-resin-bound) (>5 mmol/g, 150 mg). After being
stirred at room temperature for 16 hr the mixture was filtered
through celite (pre-washed with MeOH), and concentrated under
reduced pressure. Residue was triturated with diethyl ether and
hexanes. Resulting solid material, MeVal-Cit-PAB-OH, was suspended
in CH.sub.2Cl.sub.2 (20 mL), and to it was added MC-OSu (0.28 g,
0.896 mmol), DIEA (0.17 mL, 0.99 mmol), and DMF (15 mL). This
suspension was stirred for 16 hr, but HPLC analysis of the reaction
mixture indicated incomplete reaction, so the suspension was
concentrated under reduced pressure to a volume of 6 mL, then a 10%
NaHCO.sub.3 (aq) solution was added and the suspension stirred for
an additional 16 hr. Solvent was removed under reduced pressure,
and the residue was purified by flash column chromatography on
silica gel using a 0-10% MeOH/CH.sub.2Cl.sub.2 gradient, resulting
in 42 mg (0.072 mmol, 8% yield) of MC-MeVal-Cit-PAB-OH.
To a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol) and
bis(nitrophenyl)carbonate (2.59 g, 8.52 mmol) in CH.sub.2Cl.sub.2
(10 mL) was added DIEA (1.06 mL, 6.06 mmol). This suspension was
stirred for 5.5 hr, concentrated under reduced pressure and
purified by trituration with diethyl ether.
MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) was suspended in a
1:5 pyridine/DMF solution (3 mL), and to it was added HOBt (5 mg,
0.039 mmol), DIEA (0.17 mL, 0.978 mmol) and MMAF (compound 2, 150
mg, 0.205 mmol). This reaction mixture was stirred for 16 hr at
room temperature, and then purified by preparatory RP-HPLC
(.times.3), using a Phenomenex C.sub.12 Synergi Max-RP 80 .ANG.
Column (250.times.21.20 mm). Eluent: linear gradient 10% to 90%
MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90% MeCN/0.05%
TFA (aq) for an additional 20 minutes. MC-MeVal-Cit-PAB-MMAF (16)
was obtained as a yellowish solid (24.5 mg, 0.0182, 0.45% yield).
ES-MS m/z 1344.95 [M+H].sup.+; 1366.94 [M+Na].sup.+.
Example 23c
Preparation of Succinimide Ester of suberyl-Val-Cit-PAB-MMAF
(17)
##STR00064##
Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57
mmol, 1.5 eq.) were suspended in anhydrous pyridine, 5 mL. HOBt (10
mg, 0.076 mmol, 0.2 eq.) was added followed by DIEA (199 .mu.l,
1.14 mmol, 3 eq.). Reaction mixture was sonicated for 10 min, and
then stirred overnight at room temperature. Pyridine was removed
under reduced pressure, residue was re-suspended in
CH.sub.2Cl.sub.2. Mixture was separated by silica gel flash
chromatography in a step gradient of MeOH, from 0 to 10%, in
CH.sub.2Cl.sub.2.Product containing fractions were pulled,
concentrated, dried in vacuum overnight to give 317 mg (59% yield)
of Fmoc-Val-Cit-PAB-MMAF-OtBu. ES-MS m/z 1415.8 [M+H H].sup.+.
Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20%
TFA/CH.sub.2Cl.sub.2 (10 mL), for 2 hrs. Mixture was diluted with
CH.sub.2Cl.sub.2 (50 mL). Organic layer was washed successively
with water (2.times.30 mL) and brine (1.times.30 mL). Organic phase
was concentrated, loaded onto pad of silica gel in 10%
MeOH/CH.sub.2Cl.sub.2. Product was eluted with 30%
MeOH/CH.sub.2Cl.sub.2. After drying in vacuum overnight,
Fmoc-Val-Cit-PAB-MMAF was obtained as a white solid, 38 mg, 40%
yield. ES-MS m/z 1357.7 [M-H].sup.-.
Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in CH.sub.2Cl.sub.2 (2
mL) diethylamine (2 mL) and DMF (2 mL). Mixture was stirred for 2
hrs at room temperature. Solvent was removed under reduced
pressure. Residue was co-evaporated with pyridine (2 mL), then with
toluene (2.times.5 mL), dried in vacuum. Val-Cit-PAB-MMAF was
obtained as brownish oil, and used without further
purification.
All Val-Cit-PAB-MMAF prepared from 67 mg of Fmoc-Val-Cit-PAB-MMAF,
was suspended in pyridine (2 mL), and added to a solution of
disuccinimidyl suberate (74 mg, 0.2 mmol, 4 eq.), in pyridine (1
mL). Reaction mixture was stirred at room temperature. After 3 hrs
ether (20 mL) was added. Precipitate was collected, washed with
additional amount of ether. Reddish solid was suspended in 30%
MeOH/CH.sub.2Cl.sub.2, filtered trough a pad of silica gel with 30%
MeOH/CH.sub.2Cl.sub.2 as an eluent. Compound 17 was obtained as
white solid, 20 mg (29% yield). ES-MS m/z 1388.5 [M-H].sup.-
Example 24
In Vivo Efficacy of mcMMAF Antibody-Drug Conjugates
Efficacy of cAC10-mcMMAF in Karpas-299 ALCL Xenografts:
To evaluate the in vivo efficacy of cAC10-mcMMAF with an average of
4 drug moieties per antibody (cAC10-mcF4), Karpas-299 human ALCL
cells were implanted subcutaneously into immunodeficient C.B-17
SCID mice (5.times.10.sup.6 cells per mouse). Tumor volumes were
calculated using the formula (0.5.times.L.times.W.sup.2) where L
and W are the longer and shorter of two bidirectional measurements.
When the average tumor volume in the study animals reached
approximately 100 mm.sup.3 (range 48-162) the mice were divided
into 3 groups (5 mice per group) and were either left untreated or
were given a single intravenous injection through the tail vein of
either 1 or 2 mg/kg cAC10-mcF4 (FIG. 1). The tumors in the
untreated mice grew rapidly to an average volume of >1,000
mm.sup.3 within 7 days of the start of therapy. In contrast, all of
the cAC10-mcF4 treated tumor showed rapid regression with 3/5 in
the 1 mg/kg group and 5/5 in the 2 mg/kg group obtaining complete
tumor response. While the tumor in one of the complete responders
in the 2 mg/kg group did recur approximately 4 weeks later, there
were no detectable tumors in the remaining 4/5 responders in this
group and in the 3 complete responders in the 1 mg/kg group at 10
weeks post therapy.
Efficacy of cBR96-mcMMAF in L2987 NSCLC Xenografts:
cBR96 is a chimeric antibody that recognizes the Le.sup.Y antigen.
To evaluate the in vivo efficacy of cBR96-mcMMAF with 4 drugs per
antibody (cBR96-mcF4) L2987 non-small cell lung cancer (NSCLC)
tumor fragments were implanted into athymic nude mice. When the
tumors averaged approximately 100 mm.sup.3 the mice were divided
into 3 groups: untreated and 2 therapy groups. For therapy, as
shown in FIG. 3a, mice were administered cBR96-mcF4 at either 3 or
10 mg/kg/injection every 4 days for a total of 4 injections
(q4dx4). As shown in FIG. 3b, mice were administered cBR96-mcF4 or
a non-binding control conjugate, cAC10-mcF4, at 10 mg/kg/injection
every 4 days for a total of 4 injections (q4dx4). As shown in FIGS.
3a and 3b, BR96-mcF4 produced pronounced tumor growth delay
compared to the controls.
FIG. 2 shows an in vivo, single dose, efficacy assay of
cAC10-mcMMAF in subcutaneous L540CY. For this study there were 4
mice in the untreated group and 10 in each of the treatment
groups.
Example 25
In Vitro Efficacy of MC-MMAF Antibody-Drug Conjugates
Activity of cAC10-Antibody-Drug Conjugates Against CD30.sup.+ Cell
Lines.
FIGS. 4a and 16b show dose-response curves from a representative
experiment where cultures of Karpas 299 (anaplastic large cell
lymphoma) and L428 (Hodgkin's Lymphoma) were incubated with
serially diluted cAC10-mcMMAF (FIG. 4a) or cAC10-vcMMAF (FIG. 4b)
for 96 hours. The cultures were labeled for 4 hours with 50 .mu.M
resazurin [7-hydroxy-3H-phenoxazin-3-one 10-oxide] and the
fluorescence measured. The data were reduced in GraphPad Prism
version 4.00 using the 4-parameter dose-response curve fit
procedure. IC.sub.50 values are defined as the concentration where
growth is reduced 50% compared with untreated control cultures.
Each concentration was tested in quadruplicate.
Activity of cBR96-Antibody-Drug Conjugates Against Le.sup.v+ Cell
Lines.
FIGS. 5a and 5b show dose-response curves from a representative
experiment where cultures of H3396 (breast carcinoma) and L2987
(non small cell lung carcinoma) were incubated with serially
diluted cBR96-mcMMAF (FIG. 5a) or -vcMMAF (FIG. 5b) for 96 hours.
The cultures were labeled for 4 hours with 50 .mu.M resazurin and
the fluorescence measured. The data were reduced in GraphPad Prism
version 4.00 using the 4-parameter dose-response curve fit
procedure. IC.sub.50 values are defined as the concentration where
growth is reduced 50% compared with untreated control cultures.
Each concentration is tested in quadruplicate.
Activity of clF6-antibody-drug conjugates against CD70.sup.+ renal
cell carcinoma cell lines. FIGS. 6a and 6b show dose-response
curves from a representative experiment where cultures of Caki-1
and 786-O cells were incubated with serially diluted c1F6-mcMMAF
(FIG. 6a) or -vcMMAF (FIG. 6b) for 96 hours. The cultures were
labeled for 4 hours with 50 .mu.M resazurin and the fluorescence
measured. The data were reduced in GraphPad Prism version 4.00
using the 4-parameter dose-response curve fit procedure. IC.sub.50
values are defined as the concentration where growth is reduced 50%
compared with untreated control cultures. Each concentration is
tested in quadruplicate.
Example 26
Purification of Trastuzumab
One vial containing 440 mg HERCEPTIN.RTM. (huMAb4D5-8, rhuMAb HER2,
U.S. Pat. No. 5,821,337) antibody) was dissolved in 50 mL MES
buffer (25 mM MES, 50 mM NaCl, pH 5.6) and loaded on a cation
exchange column (Sepharose S, 15 cm.times.1.7 cm) that had been
equilibrated in the same buffer. The column was then washed with
the same buffer (5 column volumes). Trastuzumab was eluted by
raising the NaCl concentration of the buffer to 200 mM. Fractions
containing the antibody were pooled, diluted to 10 mg/mL, and
dialyzed into a buffer containing 50 mm potassium phosphate, 50 mM
NaCl, 2 mM EDTA, pH 6.5.
Example 27
Preparation of trastuzumab-MC-MMAE by Conjugation of Trastuzumab
and MC-MMAE
Trastuzumab, dissolved in 500 mM sodium borate and 500 mM sodium
chloride at pH 8.0 is treated with an excess of 100 mM
dithiothreitol (DTT). After incubation at 37.degree. C. for about
30 minutes, the buffer is exchanged by elution over Sephadex G25
resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is
checked by determining the reduced antibody concentration from the
absorbance at 280 nm of the solution and the thiol concentration by
reaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of
the absorbance at 412 nm. The reduced antibody dissolved in PBS is
chilled on ice.
The drug linker reagent, maleimidocaproyl-monomethyl auristatin E
(MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile
and water at known concentration, and added to the chilled reduced
antibody trastuzumab in PBS. After about one hour, an excess of
maleimide is added to quench the reaction and cap any unreacted
antibody thiol groups. The reaction mixture is concentrated by
centrifugal ultrafiltration and trastuzumab-MC-MMAE is purified and
desalted by elution through G25 resin in PBS, filtered through 0.2
.mu.m filters under sterile conditions, and frozen for storage.
Example 28
Preparation of trastuzumab-MC-MMAF by Conjugation of Trastuzumab
and MC-MMAF
Trastuzumab-MC-MMAF was prepared by conjugation of trastuzumab and
MC-MMAF following the procedure of Example 27.
Example 29
Preparation of trastuzumab-MC-val-cit-PAB-MMAE by Conjugation of
Trastuzumab and MC-val-cit-PAB-MMAE
Trastuzumab-MC-val-cit-PAB-MMAE was prepared by conjugation of
trastuzumab and MC-val-cit-PAB-MMAE following the procedure of
Example 27.
Example 30
Preparation of trastuzumab-MC-val-cit-PAB-MMAF by Conjugation of
Trastuzumab and MC-val-cit-PAB-MMAF 9
Trastuzumab-MC-val-cit-PAB-MMAF was prepared by conjugation of
trastuzumab and MC-val-cit-PAB-MMAF 9 following the procedure of
Example 27.
Example 31
Rat Toxicity
The acute toxicity profile of free drugs and ADC was evaluated in
adolescent Sprague-Dawley rats (75-125 gms each, Charles River
Laboratories (Hollister, Calif.). Animals were injected on day 1,
complete chemistry and hematology profiles were obtained at
baseline, day 3 and day 5 and a complete necropsy was performed on
day 5. Liver enzyme measurements was done on all animals and
routine histology as performed on three random animals for each
group for the following tissues: sternum, liver, kidney, thymus,
spleen, large and small intestine. The experimental groups were as
follows:
TABLE-US-00049 .mu.g MMAF/ MMAF/ N/ Group Administered mg/kg
m.sup.2 MAb Sex 1 Vehicle 0 0 0 2/F 2 trastuzumab-MC-val-cit- 9.94
840 4.2 6/F MMAF 3 trastuzumab-MC-val-cit- 24.90 2105 4.2 6/F MMAF
4 trastuzumab-MC(Me)-val- 10.69 840 3.9 6/F cit-PAB-MMAF 5
trastuzumab-MC(Me)-val- 26.78 2105 3.9 6/F cit-PAB-MMAF 6
trastuzumab-MC-MMAF 10.17 840 4.1 6/F 7 trastuzumab-MC-MMAF 25.50
2105 4.1 6/F 8 trastuzumab-MC-val-cit- 21.85 2105 4.8 6/F
PAB-MMAF
For trastuzumab-MC-val-cit-MMAF,
trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and
trastuzumab-MC-val-cit-PAB-MMAF, the .mu.g MMAF/m.sup.2 was
calculated using 731.5 as the MW of MMAF and 145167 as the MW of
Herceptin.
The body surface area was calculated as follows: [{(body weight in
grams to 0.667 power).times.11.8}/10000]. (Guidance for Industry
and Reviewers, 2002).
The dose solutions were administered by a single intravenous bolus
tail-vein injection on Study Day 1 at a dose volume of 10 mL/kg.
Body weights of the animals were measured pre-dose on Study Day 1
and daily thereafter. Whole blood was collected into EDTA
containing tubes for hematology analysis. Whole blood was collected
into serum separator tubes for clinical chemistry analysis. Blood
samples were collected pre-dose on Study Day-4, Study Day 3 and
Study Day 5. Whole blood was also collected into sodium heparin
containing tubes at necropsy and the plasma was frozen at
-70.degree. C. for possible later analysis. The following tissues
were collected and placed in neutral buffered formalin at necropsy:
liver, kidneys, heart, thymus, spleen, brain, sternum and sections
of the GI tract, including stomach, large and small intestine.
Sternum, small intestine, large intestine, liver, thymus, spleen
and kidney were examined.
Liver associated serum enzyme levels at each timepoint were
compared to a range (5th and 95th percentile) from normal female
Sprague-Dawley rats. White blood cell and platelet counts at each
timepoint were compared to a range (5th and 95th percentile) from
normal female Sprague-Dawley rats.
High dose study in normal female Sprague-Dawley rats:
TABLE-US-00050 Group 1: Vehicle Group 2: trastuzumab-MC-MMAF, 52.24
mg/kg, 4210 .mu.g/m.sup.2 Group 3: trastuzumab-MC-MMAF, 68.25
mg/kg, 5500 .mu.g/m.sup.2 Group 4: trastuzumab-MC-MMAF, 86.00
mg/kg, 6930 .mu.g/m.sup.2
Tissues from 11 animals were submitted for routine histology. These
animals had been part of an acute dose-ranging toxicity study using
a trastuzumab-MC-MMAF immunoconjugate. Animals were followed for 12
days following dosing.
Example 32
Cynomolgus Monkey Toxicity/Safety
Three groups of four (2 male, 2 female) naive Macaca fascicularis
(cynomolgus monkey) were studied for trastuzumab-MC-vc-PAB-MMAE and
trastuzumab-MC-vc-PAB-MMAF. Intravenous administration was
conducted at days 1 and 22 of the studies.
TABLE-US-00051 Sample Group Dose Vehicle 1 day 1 1M/1F day 22
H-MC-vc-PAB-MMAE 2 180 .mu.g/m.sup.2 (0.5 mg/kg) at day 1 2M/2F
1100 .mu.g/m.sup.2 (3.0 mg/kg) at day 22 H-MC-vc-PAB-MMAE 3 550
.mu.g/m.sup.2 (1.5 mg/kg) at day 8 2M/2F 550 .mu.g/m.sup.2 (1.5
mg/kg) at day 29 H-MC-vc-PAB-MMAE 4 880 .mu.g/m.sup.2 (2.5 mg/kg)
at day 15 2M/2F 880 .mu.g/m.sup.2 (2.5 mg/kg) at day 36 Vehicle 1
day 1 1M/1F day 22 H-MC-vc-PAB-MMAF 2 180 .mu.g/m.sup.2 (0.5 mg/kg)
at day 1 2M/2F 1100 .mu.g/m.sup.2 (3.0 mg/kg) at day 22
H-MC-vc-PAB-MMAF 3 550 .mu.g/m.sup.2 (1.5 mg/kg) at day 1 2M/2F 550
.mu.g/m.sup.2 (1.5 mg/kg) at day 22 H-MC-vc-PAB-MMAF 4 880
.mu.g/m.sup.2 (2.5 mg/kg) at day 1 2M/2F 880 .mu.g/m.sup.2 (2.5
mg/kg) at day 22 H = trastuzumab
Dosing is expressed in surface area of an animal so as to be
relevant to other species, i.e. dosage at .mu.g/m.sup.2 is
independent of species and thus comparable between species.
Formulations of ADC contained PBS, 5.4 mM sodium phosphate, 4.2 mM
potassium phosphate, 140 mM sodium chloride, pH 6.5.
Blood was collected for hematology analysis predose, and at 5 min.,
6 hr, 10 hr, and 1, 3, 5, 7, 14, 21 days after each dose.
Erythrocyte (RBC) and platelet (PLT) counts were measured by the
light scattering method. Leukocyte (WBC) count was measured by the
peroxidase/basophil method. Reticulocyte count was measured by the
light scattering method with cationic dye. Cell counts were
measured on an Advia 120 apparatus. ALT (alanine aminotransferase)
and AST (aspartate aminotransferase) were measured in U/L by
UV/NADH; IFCC methodology on an Olympus AU400 apparatus, and using
Total Ab ELISA-ECD/GxhuFc-HRP. Conj. Ab
ELISA-MMAE/MMAF//ECD-Bio/SA-HRP tests.
Example 33
Production, Characterization and Humanization of Anti-ErbB2
Monoclonal Antibody 4D5
The murine monoclonal antibody 4D5 which specifically binds the
extracellular domain of ErbB2 was produced as described in Fendly
et al. (1990) Cancer Research 50:1550-1558. Briefly, NIH
3T3/HER2-3.sub.400 cells (expressing approximately 1.times.10.sup.5
ErbB2 molecules/cell) produced as described in Hudziak et al. Proc.
Natl. Acad. Sci. (USA) 84:7158-7163 (1987) were harvested with
phosphate buffered saline (PBS) containing 25 mM EDTA and used to
immunize BALB/c mice. The mice were given injections i.p. of
10.sup.7 cells in 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice with
antisera that immunoprecipitated .sup.32P-labeled ErbB2 were given
i.p. injections of a wheat germ agglutinin-Sepharose (WGA) purified
ErbB2 membrane extract on weeks 9 and 13. This was followed by an
i.v. injection of 0.1 ml of the ErbB2 preparation and the
splenocytes were fused with mouse myeloma line X63-Ag8.653.
Hybridoma supernatants were screened for ErbB2-binding by ELISA and
radioimmunoprecipitation.
Epitope Mapping and Characterization
The ErbB2 epitope bound by monoclonal antibody 4D5 was determined
by competitive binding analysis (Fendly et al. Cancer Research
50:1550-1558 (1990)). Cross-blocking studies were done by direct
fluorescence on intact cells using the PANDEX.TM. Screen Machine to
quantitate fluorescence. The monoclonal antibody was conjugated
with fluorescein isothiocyanate (FITC), using established
procedures (Wofsy et al. Selected Methods in Cellular Immunology,
p. 287, Mishel and Schiigi (eds.) San Francisco: W.J. Freeman Co.
(1980)). Confluent monolayers of NIH 3T3/HER2-3.sub.400 cells were
trypsinized, washed once, and resuspended at 1.75.times.10.sup.6
cell/ml in cold PBS containing 0.5% bovine serum albumin (BSA) and
0.1% NaN.sub.3. A final concentration of 1% latex particles (IDC,
Portland, Oreg.) was added to reduce clogging of the PANDEX.TM.
plate membranes. Cells in suspension, 20 .mu.l, and 20 .mu.l of
purified monoclonal antibodies (100 .mu.g/ml to 0.1 .mu.g/ml) were
added to the PANDEX.TM. plate wells and incubated on ice for 30
minutes. A predetermined dilution of the FITC-labeled monoclonal
antibody in 20 .mu.l was added to each well, incubated for 30
minutes, washed, and the fluorescence was quantitated by the
PANDEX.TM.. Monoclonal antibodies were considered to share an
epitope if each blocked binding of the other by 50% or greater in
comparison to an irrelevant monoclonal antibody control. In this
experiment, monoclonal antibody 4D5 was assigned epitope I (amino
acid residues from about 529 to about 625, inclusive within the
ErbB2 extracellular domain.
The growth inhibitory characteristics of monoclonal antibody 4D5
were evaluated using the breast tumor cell line, SK-BR-3 (see
Hudziak et al. (1989) Molec. Cell. Biol. 9(3):1165-1172). Briefly,
SK-BR-3 cells were detached by using 0.25% (vol/vol) trypsin and
suspended in complete medium at a density of 4.times.10.sup.5 cells
per ml. Aliquots of 100 .mu.l (4.times.10.sup.4 cells) were plated
into 96-well microdilution plates, the cells were allowed to
adhere, and 100 .mu.l of media alone or media containing monoclonal
antibody (final concentration 5 .mu.g/ml) was then added. After 72
hours, plates were washed twice with PBS (pH 7.5), stained with
crystal violet (0.5% in methanol), and analyzed for relative cell
proliferation as described in Sugarman et al. (1985) Science
230:943-945. Monoclonal antibody 4D5 inhibited SK-BR-3 relative
cell proliferation by about 56%.
Monoclonal antibody 4D5 was also evaluated for its ability to
inhibit HRG-stimulated tyrosine phosphorylation of proteins in the
M.sub.r. 180,000 range from whole-cell lysates of MCF7 cells (Lewis
et al. (1996) Cancer Research 56:1457-1465). MCF7 cells are
reported to express all known ErbB receptors, but at relatively low
levels. Since ErbB2, ErbB3, and ErbB4 have nearly identical
molecular sizes, it is not possible to discern which protein is
becoming tyrosine phosphorylated when whole-cell lysates are
evaluated by Western blot analysis. However, these cells are ideal
for HRG tyrosine phosphorylation assays because under the assay
conditions used, in the absence of exogenously added HRG, they
exhibit low to undetectable levels of tyrosine phosphorylation
proteins in the M.sub.r 180,000 range.
MCF7 cells were plated in 24-well plates and monoclonal antibodies
to ErbB2 were added to each well and incubated for 30 minutes at
room temperature; then rHRG.beta.1.sub.177-244 was added to each
well to a final concentration of 0.2 nM, and the incubation was
continued for 8 minutes. Media was carefully aspirated from each
well, and reactions were stopped by the addition of 100 .mu.l of
SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8).
Each sample (25 .mu.l) was electrophoresed on a 4-12% gradient gel
(Novex) and then electrophoretically transferred to polyvinylidene
difluoride membrane. Antiphosphotyrosine (4G10, from UBI, used at 1
.mu.g/ml) immunoblots were developed, and the intensity of the
predominant reactive band at M.sub.r180,000 was quantified by
reflectance densitometry, as described previously (Holmes et al.
(1992) Science 256:1205-1210; Sliwkowski et al. J. Biol. Chem.
269:14661-14665 (1994)).
Monoclonal antibody 4D5 significantly inhibited the generation of a
HRG-induced tyrosine phosphorylation signal at M.sub.r 180,000. In
the absence of HRG, but was unable to stimulate tyrosine
phosphorylation of proteins in the M.sub.r 180,000 range. Also,
this antibody does not cross-react with EGFR (Fendly et al. Cancer
Research 50:1550-1558 (1990)), ErbB3, or ErbB4. Monoclonal antibody
4D5 was able to block HRG stimulation of tyrosine phosphorylation
by 50%.
The growth inhibitory effect of monoclonal antibody 4D5 on
MDA-MB-175 and SK-BR-3 cells in the presence or absence of
exogenous rHRG.beta.1 was assessed (Schaefer et al. Oncogene
15:1385-1394 (1997)). ErbB2 levels in MDA-MB-175 cells are 4-6
times higher than the level found in normal breast epithelial cells
and the ErbB2-ErbB4 receptor is constitutively tyrosine
phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 was
able to inhibit cell proliferation of MDA-MB-175 cells, both in the
presence and absence of exogenous HRG. Inhibition of cell
proliferation by 4D5 is dependent on the ErbB2 expression level
(Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993)). A
maximum inhibition of 66% in SK-BR-3 cells could be detected.
However this effect could be overcome by exogenous HRG.
The murine monoclonal antibody 4D5 was humanized, using a "gene
conversion mutagenesis" strategy, as described in U.S. Pat. No.
5,821,337, the entire disclosure of which is hereby expressly
incorporated by reference. The humanized monoclonal antibody 4D5
used in the following experiments is designated huMAb4D5-8. This
antibody is of IgG1 isotype.
REFERENCES CITED
The present invention is not to be limited in scope by the specific
embodiments disclosed in the examples which are intended as
illustrations of a few aspects of the invention and any embodiments
that are functionally equivalent are within the scope of this
invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art and are intended to fall within the
scope of the appended claims.
All references cited herein are incorporated by reference in their
entirety and for all purposes to the same extent as if each
individual publication or patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
SEQUENCE LISTINGS
1
351502PRTHomo sapiensBMPR1B 1Met Leu Leu Arg Ser Ala Gly Lys Leu
Asn Val Gly Thr Lys Lys Glu1 5 10 15Asp Gly Glu Ser Thr Ala Pro Thr
Pro Arg Pro Lys Val Leu Arg Cys 20 25 30Lys Cys His His His Cys Pro
Glu Asp Ser Val Asn Asn Ile Cys Ser 35 40 45Thr Asp Gly Tyr Cys Phe
Thr Met Ile Glu Glu Asp Asp Ser Gly Leu 50 55 60Pro Val Val Thr Ser
Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln65 70 75 80Cys Arg Asp
Thr Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 85 90 95Thr Glu
Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro 100 105
110Leu Lys Asn Arg Asp Phe Val Asp Gly Pro Ile His His Arg Ala Leu
115 120 125Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile
Ile Leu 130 135 140Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Thr Arg
Pro Arg Tyr Ser145 150 155 160Ile Gly Leu Glu Gln Asp Glu Thr Tyr
Ile Pro Pro Gly Glu Ser Leu 165 170 175Arg Asp Leu Ile Glu Gln Ser
Gln Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190Pro Leu Leu Val Gln
Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys 195 200 205Gln Ile Gly
Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 210 215 220Gly
Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser225 230
235 240Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His
Glu 245 250 255Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr
Gly Ser Trp 260 265 270Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu
Asn Gly Ser Leu Tyr 275 280 285Asp Tyr Leu Lys Ser Thr Thr Leu Asp
Ala Lys Ser Met Leu Lys Leu 290 295 300Ala Tyr Ser Ser Val Ser Gly
Leu Cys His Leu His Thr Glu Ile Phe305 310 315 320Ser Thr Gln Gly
Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325 330 335Asn Ile
Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly 340 345
350Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro
355 360 365Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val
Leu Asp 370 375 380Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile
Met Ala Asp Met385 390 395 400Tyr Ser Phe Gly Leu Ile Leu Trp Glu
Val Ala Arg Arg Cys Val Ser 405 410 415Gly Gly Ile Val Glu Glu Tyr
Gln Leu Pro Tyr His Asp Leu Val Pro 420 425 430Ser Asp Pro Ser Tyr
Glu Asp Met Arg Glu Ile Val Cys Ile Lys Lys 435 440 445Leu Arg Pro
Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg 450 455 460Gln
Met Gly Lys Leu Met Thr Glu Cys Trp Ala His Asn Pro Ala Ser465 470
475 480Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser
Glu 485 490 495Ser Gln Asp Ile Lys Leu 5002507PRTHomo sapiensSLC7A5
2Met Ala Gly Ala Gly Pro Lys Arg Arg Ala Leu Ala Ala Pro Ala Ala1 5
10 15Glu Glu Lys Glu Glu Ala Arg Glu Lys Met Leu Ala Ala Lys Ser
Ala 20 25 30Asp Gly Ser Ala Pro Ala Gly Glu Gly Glu Gly Val Thr Leu
Gln Arg 35 40 45Asn Ile Thr Leu Leu Asn Gly Val Ala Ile Ile Val Gly
Thr Ile Ile 50 55 60Gly Ser Gly Ile Phe Val Thr Pro Thr Gly Val Leu
Lys Glu Ala Gly65 70 75 80Ser Pro Gly Leu Ala Leu Val Val Trp Ala
Ala Cys Gly Val Phe Ser 85 90 95Ile Val Gly Ala Leu Cys Tyr Ala Glu
Leu Gly Thr Thr Ile Ser Lys 100 105 110Ser Gly Gly Asp Tyr Ala Tyr
Met Leu Glu Val Tyr Gly Ser Leu Pro 115 120 125Ala Phe Leu Lys Leu
Trp Ile Glu Leu Leu Ile Ile Arg Pro Ser Ser 130 135 140Gln Tyr Ile
Val Ala Leu Val Phe Ala Thr Tyr Leu Leu Lys Pro Leu145 150 155
160Phe Pro Thr Cys Pro Val Pro Glu Glu Ala Ala Lys Leu Val Ala Cys
165 170 175Leu Cys Val Leu Leu Leu Thr Ala Val Asn Cys Tyr Ser Val
Lys Ala 180 185 190Ala Thr Arg Val Gln Asp Ala Phe Ala Ala Ala Lys
Leu Leu Ala Leu 195 200 205Ala Leu Ile Ile Leu Leu Gly Phe Val Gln
Ile Gly Lys Gly Val Val 210 215 220Ser Asn Leu Asp Pro Asn Phe Ser
Phe Glu Gly Thr Lys Leu Asp Val225 230 235 240Gly Asn Ile Val Leu
Ala Leu Tyr Ser Gly Leu Phe Ala Tyr Gly Gly 245 250 255Trp Asn Tyr
Leu Asn Phe Val Thr Glu Glu Met Ile Asn Pro Tyr Arg 260 265 270Asn
Leu Pro Leu Ala Ile Ile Ile Ser Leu Pro Ile Val Thr Leu Val 275 280
285Tyr Val Leu Thr Asn Leu Ala Tyr Phe Thr Thr Leu Ser Thr Glu Gln
290 295 300Met Leu Ser Ser Glu Ala Val Ala Val Asp Phe Gly Asn Tyr
His Leu305 310 315 320Gly Val Met Ser Trp Ile Ile Pro Val Phe Val
Gly Leu Ser Cys Phe 325 330 335Gly Ser Val Asn Gly Ser Leu Phe Thr
Ser Ser Arg Leu Phe Phe Val 340 345 350Gly Ser Arg Glu Gly His Leu
Pro Ser Ile Leu Ser Met Ile His Pro 355 360 365Gln Leu Leu Thr Pro
Val Pro Ser Leu Val Phe Thr Cys Val Met Thr 370 375 380Leu Leu Tyr
Ala Phe Ser Lys Asp Ile Phe Ser Val Ile Asn Phe Phe385 390 395
400Ser Phe Phe Asn Trp Leu Cys Val Ala Leu Ala Ile Ile Gly Met Ile
405 410 415Trp Leu Arg His Arg Lys Pro Glu Leu Glu Arg Pro Ile Lys
Val Asn 420 425 430Leu Ala Leu Pro Val Phe Phe Ile Leu Ala Cys Leu
Phe Leu Ile Ala 435 440 445Val Ser Phe Trp Lys Thr Pro Val Glu Cys
Gly Ile Gly Phe Thr Ile 450 455 460Ile Leu Ser Gly Leu Pro Val Tyr
Phe Phe Gly Val Trp Trp Lys Asn465 470 475 480Lys Pro Lys Trp Leu
Leu Gln Gly Ile Phe Ser Thr Thr Val Leu Cys 485 490 495Gln Lys Leu
Met Gln Val Val Pro Gln Glu Thr 500 5053339PRTHomo sapiensSTEAP1
3Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met1 5
10 15Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp
Thr 20 25 30Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu
His Gln 35 40 45Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu
Gln His Thr 50 55 60Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys
Ile Ala Ala Ile65 70 75 80Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu
Leu Arg Glu Val Ile His 85 90 95Pro Leu Ala Thr Ser His Gln Gln Tyr
Phe Tyr Lys Ile Pro Ile Leu 100 105 110Val Ile Asn Lys Val Leu Pro
Met Val Ser Ile Thr Leu Leu Ala Leu 115 120 125Val Tyr Leu Pro Gly
Val Ile Ala Ala Ile Val Gln Leu His Asn Gly 130 135 140Thr Lys Tyr
Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr145 150 155
160Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala
165 170 175Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr
Lys Leu 180 185 190Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys
Glu Asp Ala Trp 195 200 205Ile Glu His Asp Val Trp Arg Met Glu Ile
Tyr Val Ser Leu Gly Ile 210 215 220Val Gly Leu Ala Ile Leu Ala Leu
Leu Ala Val Thr Ser Ile Pro Ser225 230 235 240Val Ser Asp Ser Leu
Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys 245 250 255Leu Gly Ile
Val Ser Leu Leu Leu Gly Thr Ile His Ala Leu Ile Phe 260 265 270Ala
Trp Asn Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro 275 280
285Pro Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile Phe
290 295 300Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu
Lys Ile305 310 315 320Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn
Lys Thr Glu Ile Cys 325 330 335Ser Gln Leu46995PRTHomo sapiensMUC16
4Pro Val Thr Ser Leu Leu Thr Pro Gly Leu Val Ile Thr Thr Asp Arg1 5
10 15Met Gly Ile Ser Arg Glu Pro Gly Thr Ser Ser Thr Ser Asn Leu
Ser 20 25 30Ser Thr Ser His Glu Arg Leu Thr Thr Leu Glu Asp Thr Val
Asp Thr 35 40 45Glu Ala Met Gln Pro Ser Thr His Thr Ala Val Thr Asn
Val Arg Thr 50 55 60Ser Ile Ser Gly His Glu Ser Gln Ser Ser Val Leu
Ser Asp Ser Glu65 70 75 80Thr Pro Lys Ala Thr Ser Pro Met Gly Thr
Thr Tyr Thr Met Gly Glu 85 90 95Thr Ser Val Ser Ile Ser Thr Ser Asp
Phe Phe Glu Thr Ser Arg Ile 100 105 110Gln Ile Glu Pro Thr Ser Ser
Leu Thr Ser Gly Leu Arg Glu Thr Ser 115 120 125Ser Ser Glu Arg Ile
Ser Ser Ala Thr Glu Gly Ser Thr Val Leu Ser 130 135 140Glu Val Pro
Ser Gly Ala Thr Thr Glu Val Ser Arg Thr Glu Val Ile145 150 155
160Ser Ser Arg Gly Thr Ser Met Ser Gly Pro Asp Gln Phe Thr Ile Ser
165 170 175Pro Asp Ile Ser Thr Glu Ala Ile Thr Arg Leu Ser Thr Ser
Pro Ile 180 185 190Met Thr Glu Ser Ala Glu Ser Ala Ile Thr Ile Glu
Thr Gly Ser Pro 195 200 205Gly Ala Thr Ser Glu Gly Thr Leu Thr Leu
Asp Thr Ser Thr Thr Thr 210 215 220Phe Trp Ser Gly Thr His Ser Thr
Ala Ser Pro Gly Phe Ser His Ser225 230 235 240Glu Met Thr Thr Leu
Met Ser Arg Thr Pro Gly Asp Val Pro Trp Pro 245 250 255Ser Leu Pro
Ser Val Glu Glu Ala Ser Ser Val Ser Ser Ser Leu Ser 260 265 270Ser
Pro Ala Met Thr Ser Thr Ser Phe Phe Ser Thr Leu Pro Glu Ser 275 280
285Ile Ser Ser Ser Pro His Pro Val Thr Ala Leu Leu Thr Leu Gly Pro
290 295 300Val Lys Thr Thr Asp Met Leu Arg Thr Ser Ser Glu Pro Glu
Thr Ser305 310 315 320Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu
Ile Leu Ala Thr Ser 325 330 335Glu Val Thr Lys Asp Arg Glu Lys Ile
His Pro Ser Ser Asn Thr Pro 340 345 350Val Val Asn Val Gly Thr Val
Ile Tyr Lys His Leu Ser Pro Ser Ser 355 360 365Val Leu Ala Asp Leu
Val Thr Thr Lys Pro Thr Ser Pro Met Ala Thr 370 375 380Thr Ser Thr
Leu Gly Asn Thr Ser Val Ser Thr Ser Thr Pro Ala Phe385 390 395
400Pro Glu Thr Met Met Thr Gln Pro Thr Ser Ser Leu Thr Ser Gly Leu
405 410 415Arg Glu Ile Ser Thr Ser Gln Glu Thr Ser Ser Ala Thr Glu
Arg Ser 420 425 430Ala Ser Leu Ser Gly Met Pro Thr Gly Ala Thr Thr
Lys Val Ser Arg 435 440 445Thr Glu Ala Leu Ser Leu Gly Arg Thr Ser
Thr Pro Gly Pro Ala Gln 450 455 460Ser Thr Ile Ser Pro Glu Ile Ser
Thr Glu Thr Ile Thr Arg Ile Ser465 470 475 480Thr Pro Leu Thr Thr
Thr Gly Ser Ala Glu Met Thr Ile Thr Pro Lys 485 490 495Thr Gly His
Ser Gly Ala Ser Ser Gln Gly Thr Phe Thr Leu Asp Thr 500 505 510Ser
Ser Arg Ala Ser Trp Pro Gly Thr His Ser Ala Ala Thr His Arg 515 520
525Ser Pro His Ser Gly Met Thr Thr Pro Met Ser Arg Gly Pro Glu Asp
530 535 540Val Ser Trp Pro Ser Arg Pro Ser Val Glu Lys Thr Ser Pro
Pro Ser545 550 555 560Ser Leu Val Ser Leu Ser Ala Val Thr Ser Pro
Ser Pro Leu Tyr Ser 565 570 575Thr Pro Ser Glu Ser Ser His Ser Ser
Pro Leu Arg Val Thr Ser Leu 580 585 590Phe Thr Pro Val Met Met Lys
Thr Thr Asp Met Leu Asp Thr Ser Leu 595 600 605Glu Pro Val Thr Thr
Ser Pro Pro Ser Met Asn Ile Thr Ser Asp Glu 610 615 620Ser Leu Ala
Thr Ser Lys Ala Thr Met Glu Thr Glu Ala Ile Gln Leu625 630 635
640Ser Glu Asn Thr Ala Val Thr Gln Met Gly Thr Ile Ser Ala Arg Gln
645 650 655Glu Phe Tyr Ser Ser Tyr Pro Gly Leu Pro Glu Pro Ser Lys
Val Thr 660 665 670Ser Pro Val Val Thr Ser Ser Thr Ile Lys Asp Ile
Val Ser Thr Thr 675 680 685Ile Pro Ala Ser Ser Glu Ile Thr Arg Ile
Glu Met Glu Ser Thr Ser 690 695 700Thr Leu Thr Pro Thr Pro Arg Glu
Thr Ser Thr Ser Gln Glu Ile His705 710 715 720Ser Ala Thr Lys Pro
Ser Thr Val Pro Tyr Lys Ala Leu Thr Ser Ala 725 730 735Thr Ile Glu
Asp Ser Met Thr Gln Val Met Ser Ser Ser Arg Gly Pro 740 745 750Ser
Pro Asp Gln Ser Thr Met Ser Gln Asp Ile Ser Thr Glu Val Ile 755 760
765Thr Arg Leu Ser Thr Ser Pro Ile Lys Thr Glu Ser Thr Glu Met Thr
770 775 780Ile Thr Thr Gln Thr Gly Ser Pro Gly Ala Thr Ser Arg Gly
Thr Leu785 790 795 800Thr Leu Asp Thr Ser Thr Thr Phe Met Ser Gly
Thr His Ser Thr Ala 805 810 815Ser Gln Gly Phe Ser His Ser Gln Met
Thr Ala Leu Met Ser Arg Thr 820 825 830Pro Gly Glu Val Pro Trp Leu
Ser His Pro Ser Val Glu Glu Ala Ser 835 840 845Ser Ala Ser Phe Ser
Leu Ser Ser Pro Val Met Thr Ser Ser Ser Pro 850 855 860Val Ser Ser
Thr Leu Pro Asp Ser Ile His Ser Ser Ser Leu Pro Val865 870 875
880Thr Ser Leu Leu Thr Ser Gly Leu Val Lys Thr Thr Glu Leu Leu Gly
885 890 895Thr Ser Ser Glu Pro Glu Thr Ser Ser Pro Pro Asn Leu Ser
Ser Thr 900 905 910Ser Ala Glu Ile Leu Ala Thr Thr Glu Val Thr Thr
Asp Thr Glu Lys 915 920 925Leu Glu Met Thr Asn Val Val Thr Ser Gly
Tyr Thr His Glu Ser Pro 930 935 940Ser Ser Val Leu Ala Asp Ser Val
Thr Thr Lys Ala Thr Ser Ser Met945 950 955 960Gly Ile Thr Tyr Pro
Thr Gly Asp Thr Asn Val Leu Thr Ser Thr Pro 965 970 975Ala Phe Ser
Asp Thr Ser Arg Ile Gln Thr Lys Ser Lys Leu Ser Leu 980 985 990Thr
Pro Gly Leu Met Glu Thr Ser Ile Ser Glu Glu Thr Ser Ser Ala 995
1000 1005Thr Glu Lys Ser Thr Val Leu Ser Ser Val Pro Thr Gly Ala
Thr Thr 1010 1015 1020Glu Val Ser Arg Thr Glu Ala Ile Ser Ser Ser
Arg Thr Ser Ile Pro1025 1030 1035 1040Gly Pro Ala Gln Ser Thr Met
Ser Ser Asp Thr Ser Met Glu Thr Ile 1045 1050 1055Thr Arg Ile Ser
Thr Pro Leu Thr Arg Lys Glu Ser Thr Asp Met Ala 1060 1065 1070Ile
Thr Pro Lys Thr Gly Pro Ser Gly Ala Thr Ser Gln Gly Thr Phe 1075
1080 1085Thr Leu Asp Ser Ser Ser Thr Ala Ser Trp Pro Gly Thr His
Ser Ala 1090 1095 1100Thr Thr Gln Arg Phe Pro Arg Ser Val Val Thr
Thr Pro Met Ser Arg1105
1110 1115 1120Gly Pro Glu Asp Val Ser Trp Pro Ser Pro Leu Ser Val
Glu Lys Asn 1125 1130 1135Ser Pro Pro Ser Ser Leu Val Ser Ser Ser
Ser Val Thr Ser Pro Ser 1140 1145 1150Pro Leu Tyr Ser Thr Pro Ser
Gly Ser Ser His Ser Ser Pro Val Pro 1155 1160 1165Val Thr Ser Leu
Phe Thr Ser Ile Met Met Lys Ala Thr Asp Met Leu 1170 1175 1180Asp
Ala Ser Leu Glu Pro Glu Thr Thr Ser Ala Pro Asn Met Asn Ile1185
1190 1195 1200Thr Ser Asp Glu Ser Leu Ala Ala Ser Lys Ala Thr Thr
Glu Thr Glu 1205 1210 1215Ala Ile His Val Phe Glu Asn Thr Ala Ala
Ser His Val Glu Thr Thr 1220 1225 1230Ser Ala Thr Glu Glu Leu Tyr
Ser Ser Ser Pro Gly Phe Ser Glu Pro 1235 1240 1245Thr Lys Val Ile
Ser Pro Val Val Thr Ser Ser Ser Ile Arg Asp Asn 1250 1255 1260Met
Val Ser Thr Thr Met Pro Gly Ser Ser Gly Ile Thr Arg Ile Glu1265
1270 1275 1280Ile Glu Ser Met Ser Ser Leu Thr Pro Gly Leu Arg Glu
Thr Arg Thr 1285 1290 1295Ser Gln Asp Ile Thr Ser Ser Thr Glu Thr
Ser Thr Val Leu Tyr Lys 1300 1305 1310Met Pro Ser Gly Ala Thr Pro
Glu Val Ser Arg Thr Glu Val Met Pro 1315 1320 1325Ser Ser Arg Thr
Ser Ile Pro Gly Pro Ala Gln Ser Thr Met Ser Leu 1330 1335 1340Asp
Ile Ser Asp Glu Val Val Thr Arg Leu Ser Thr Ser Pro Ile Met1345
1350 1355 1360Thr Glu Ser Ala Glu Ile Thr Ile Thr Thr Gln Thr Gly
Tyr Ser Leu 1365 1370 1375Ala Thr Ser Gln Val Thr Leu Pro Leu Gly
Thr Ser Met Thr Phe Leu 1380 1385 1390Ser Gly Thr His Ser Thr Met
Ser Gln Gly Leu Ser His Ser Glu Met 1395 1400 1405Thr Asn Leu Met
Ser Arg Gly Pro Glu Ser Leu Ser Trp Thr Ser Pro 1410 1415 1420Arg
Phe Val Glu Thr Thr Arg Ser Ser Ser Ser Leu Thr Ser Leu Pro1425
1430 1435 1440Leu Thr Thr Ser Leu Ser Pro Val Ser Ser Thr Leu Leu
Asp Ser Ser 1445 1450 1455Pro Ser Ser Pro Leu Pro Val Thr Ser Leu
Ile Leu Pro Gly Leu Val 1460 1465 1470Lys Thr Thr Glu Val Leu Asp
Thr Ser Ser Glu Pro Lys Thr Ser Ser 1475 1480 1485Ser Pro Asn Leu
Ser Ser Thr Ser Val Glu Ile Pro Ala Thr Ser Glu 1490 1495 1500Ile
Met Thr Asp Thr Glu Lys Ile His Pro Ser Ser Asn Thr Ala Val1505
1510 1515 1520Ala Lys Val Arg Thr Ser Ser Ser Val His Glu Ser His
Ser Ser Val 1525 1530 1535Leu Ala Asp Ser Glu Thr Thr Ile Thr Ile
Pro Ser Met Gly Ile Thr 1540 1545 1550Ser Ala Val Glu Asp Thr Thr
Val Phe Thr Ser Asn Pro Ala Phe Ser 1555 1560 1565Glu Thr Arg Arg
Ile Pro Thr Glu Pro Thr Phe Ser Leu Thr Pro Gly 1570 1575 1580Phe
Arg Glu Thr Ser Thr Ser Glu Glu Thr Thr Ser Ile Thr Glu Thr1585
1590 1595 1600Ser Ala Val Leu Phe Gly Val Pro Thr Ser Ala Thr Thr
Glu Val Ser 1605 1610 1615Met Thr Glu Ile Met Ser Ser Asn Arg Thr
His Ile Pro Asp Ser Asp 1620 1625 1630Gln Ser Thr Met Ser Pro Asp
Ile Ile Thr Glu Val Ile Thr Arg Leu 1635 1640 1645Ser Ser Ser Ser
Met Met Ser Glu Ser Thr Gln Met Thr Ile Thr Thr 1650 1655 1660Gln
Lys Ser Ser Pro Gly Ala Thr Ala Gln Ser Thr Leu Thr Leu Ala1665
1670 1675 1680Thr Thr Thr Ala Pro Leu Ala Arg Thr His Ser Thr Val
Pro Pro Arg 1685 1690 1695Phe Leu His Ser Glu Met Thr Thr Leu Met
Ser Arg Ser Pro Glu Asn 1700 1705 1710Pro Ser Trp Lys Ser Ser Pro
Phe Val Glu Lys Thr Ser Ser Ser Ser 1715 1720 1725Ser Leu Leu Ser
Leu Pro Val Thr Thr Ser Pro Ser Val Ser Ser Thr 1730 1735 1740Leu
Pro Gln Ser Ile Pro Ser Ser Ser Phe Ser Val Thr Ser Leu Leu1745
1750 1755 1760Thr Pro Gly Met Val Lys Thr Thr Asp Thr Ser Thr Glu
Pro Gly Thr 1765 1770 1775Ser Leu Ser Pro Asn Leu Ser Gly Thr Ser
Val Glu Ile Leu Ala Ala 1780 1785 1790Ser Glu Val Thr Thr Asp Thr
Glu Lys Ile His Pro Ser Ser Ser Met 1795 1800 1805Ala Val Thr Asn
Val Gly Thr Thr Ser Ser Gly His Glu Leu Tyr Ser 1810 1815 1820Ser
Val Ser Ile His Ser Glu Pro Ser Lys Ala Thr Tyr Pro Val Gly1825
1830 1835 1840Thr Pro Ser Ser Met Ala Glu Thr Ser Ile Ser Thr Ser
Met Pro Ala 1845 1850 1855Asn Phe Glu Thr Thr Gly Phe Glu Ala Glu
Pro Phe Ser His Leu Thr 1860 1865 1870Ser Gly Leu Arg Lys Thr Asn
Met Ser Leu Asp Thr Ser Ser Val Thr 1875 1880 1885Pro Thr Asn Thr
Pro Ser Ser Pro Gly Ser Thr His Leu Leu Gln Ser 1890 1895 1900Ser
Lys Thr Asp Phe Thr Ser Ser Ala Lys Thr Ser Ser Pro Asp Trp1905
1910 1915 1920Pro Pro Ala Ser Gln Tyr Thr Glu Ile Pro Val Asp Ile
Ile Thr Pro 1925 1930 1935Phe Asn Ala Ser Pro Ser Ile Thr Glu Ser
Thr Gly Ile Thr Ser Phe 1940 1945 1950Pro Glu Ser Arg Phe Thr Met
Ser Val Thr Glu Ser Thr His His Leu 1955 1960 1965Ser Thr Asp Leu
Leu Pro Ser Ala Glu Thr Ile Ser Thr Gly Thr Val 1970 1975 1980Met
Pro Ser Leu Ser Glu Ala Met Thr Ser Phe Ala Thr Thr Gly Val1985
1990 1995 2000Pro Arg Ala Ile Ser Gly Ser Gly Ser Pro Phe Ser Arg
Thr Glu Ser 2005 2010 2015Gly Pro Gly Asp Ala Thr Leu Ser Thr Ile
Ala Glu Ser Leu Pro Ser 2020 2025 2030Ser Thr Pro Val Pro Phe Ser
Ser Ser Thr Phe Thr Thr Thr Asp Ser 2035 2040 2045Ser Thr Ile Pro
Ala Leu His Glu Ile Thr Ser Ser Ser Ala Thr Pro 2050 2055 2060Tyr
Arg Val Asp Thr Ser Leu Gly Thr Glu Ser Ser Thr Thr Glu Gly2065
2070 2075 2080Arg Leu Val Met Val Ser Thr Leu Asp Thr Ser Ser Gln
Pro Gly Arg 2085 2090 2095Thr Ser Ser Ser Pro Ile Leu Asp Thr Arg
Met Thr Glu Ser Val Glu 2100 2105 2110Leu Gly Thr Val Thr Ser Ala
Tyr Gln Val Pro Ser Leu Ser Thr Arg 2115 2120 2125Leu Thr Arg Thr
Asp Gly Ile Met Glu His Ile Thr Lys Ile Pro Asn 2130 2135 2140Glu
Ala Ala His Arg Gly Thr Ile Arg Pro Val Lys Gly Pro Gln Thr2145
2150 2155 2160Ser Thr Ser Pro Ala Ser Pro Lys Gly Leu His Thr Gly
Gly Thr Lys 2165 2170 2175Arg Met Glu Thr Thr Thr Thr Ala Leu Lys
Thr Thr Thr Thr Ala Leu 2180 2185 2190Lys Thr Thr Ser Arg Ala Thr
Leu Thr Thr Ser Val Tyr Thr Pro Thr 2195 2200 2205Leu Gly Thr Leu
Thr Pro Leu Asn Ala Ser Met Gln Met Ala Ser Thr 2210 2215 2220Ile
Pro Thr Glu Met Met Ile Thr Thr Pro Tyr Val Phe Pro Asp Val2225
2230 2235 2240Pro Glu Thr Thr Ser Ser Leu Ala Thr Ser Leu Gly Ala
Glu Thr Ser 2245 2250 2255Thr Ala Leu Pro Arg Thr Thr Pro Ser Val
Phe Asn Arg Glu Ser Glu 2260 2265 2270Thr Thr Ala Ser Leu Val Ser
Arg Ser Gly Ala Glu Arg Ser Pro Val 2275 2280 2285Ile Gln Thr Leu
Asp Val Ser Ser Ser Glu Pro Asp Thr Thr Ala Ser 2290 2295 2300Trp
Val Ile His Pro Ala Glu Thr Ile Pro Thr Val Ser Lys Thr Thr2305
2310 2315 2320Pro Asn Phe Phe His Ser Glu Leu Asp Thr Val Ser Ser
Thr Ala Thr 2325 2330 2335Ser His Gly Ala Asp Val Ser Ser Ala Ile
Pro Thr Asn Ile Ser Pro 2340 2345 2350Ser Glu Leu Asp Ala Leu Thr
Pro Leu Val Thr Ile Ser Gly Thr Asp 2355 2360 2365Thr Ser Thr Thr
Phe Pro Thr Leu Thr Lys Ser Pro His Glu Thr Glu 2370 2375 2380Thr
Arg Thr Thr Trp Leu Thr His Pro Ala Glu Thr Ser Ser Thr Ile2385
2390 2395 2400Pro Arg Thr Ile Pro Asn Phe Ser His His Glu Ser Asp
Ala Thr Pro 2405 2410 2415Ser Ile Ala Thr Ser Pro Gly Ala Glu Thr
Ser Ser Ala Ile Pro Ile 2420 2425 2430Met Thr Val Ser Pro Gly Ala
Glu Asp Leu Val Thr Ser Gln Val Thr 2435 2440 2445Ser Ser Gly Thr
Asp Arg Asn Met Thr Ile Pro Thr Leu Thr Leu Ser 2450 2455 2460Pro
Gly Glu Pro Lys Thr Ile Ala Ser Leu Val Thr His Pro Glu Ala2465
2470 2475 2480Gln Thr Ser Ser Ala Ile Pro Thr Ser Thr Ile Ser Pro
Ala Val Ser 2485 2490 2495Arg Leu Val Thr Ser Met Val Thr Ser Leu
Ala Ala Lys Thr Ser Thr 2500 2505 2510Thr Asn Arg Ala Leu Thr Asn
Ser Pro Gly Glu Pro Ala Thr Thr Val 2515 2520 2525Ser Leu Val Thr
His Ser Ala Gln Thr Ser Pro Thr Val Pro Trp Thr 2530 2535 2540Thr
Ser Ile Phe Phe His Ser Lys Ser Asp Thr Thr Pro Ser Met Thr2545
2550 2555 2560Thr Ser His Gly Ala Glu Ser Ser Ser Ala Val Pro Thr
Pro Thr Val 2565 2570 2575Ser Thr Glu Val Pro Gly Val Val Thr Pro
Leu Val Thr Ser Ser Arg 2580 2585 2590Ala Val Ile Ser Thr Thr Ile
Pro Ile Leu Thr Leu Ser Pro Gly Glu 2595 2600 2605Pro Glu Thr Thr
Pro Ser Met Ala Thr Ser His Gly Glu Glu Ala Ser 2610 2615 2620Ser
Ala Ile Pro Thr Pro Thr Val Ser Pro Gly Val Pro Gly Val Val2625
2630 2635 2640Thr Ser Leu Val Thr Ser Ser Arg Ala Val Thr Ser Thr
Thr Ile Pro 2645 2650 2655Ile Leu Thr Phe Ser Leu Gly Glu Pro Glu
Thr Thr Pro Ser Met Ala 2660 2665 2670Thr Ser His Gly Thr Glu Ala
Gly Ser Ala Val Pro Thr Val Leu Pro 2675 2680 2685Glu Val Pro Gly
Met Val Thr Ser Leu Val Ala Ser Ser Arg Ala Val 2690 2695 2700Thr
Ser Thr Thr Leu Pro Thr Leu Thr Leu Ser Pro Gly Glu Pro Glu2705
2710 2715 2720Thr Thr Pro Ser Met Ala Thr Ser His Gly Ala Glu Ala
Ser Ser Thr 2725 2730 2735Val Pro Thr Val Ser Pro Glu Val Pro Gly
Val Val Thr Ser Leu Val 2740 2745 2750Thr Ser Ser Ser Gly Val Asn
Ser Thr Ser Ile Pro Thr Leu Ile Leu 2755 2760 2765Ser Pro Gly Glu
Leu Glu Thr Thr Pro Ser Met Ala Thr Ser His Gly 2770 2775 2780Ala
Glu Ala Ser Ser Ala Val Pro Thr Pro Thr Val Ser Pro Gly Val2785
2790 2795 2800Ser Gly Val Val Thr Pro Leu Val Thr Ser Ser Arg Ala
Val Thr Ser 2805 2810 2815Thr Thr Ile Pro Ile Leu Thr Leu Ser Ser
Ser Glu Pro Glu Thr Thr 2820 2825 2830Pro Ser Met Ala Thr Ser His
Gly Val Glu Ala Ser Ser Ala Val Leu 2835 2840 2845Thr Val Ser Pro
Glu Val Pro Gly Met Val Thr Phe Leu Val Thr Ser 2850 2855 2860Ser
Arg Ala Val Thr Ser Thr Thr Ile Pro Thr Leu Thr Ile Ser Ser2865
2870 2875 2880Asp Glu Pro Glu Thr Thr Thr Ser Leu Val Thr His Ser
Glu Ala Lys 2885 2890 2895Met Ile Ser Ala Ile Pro Thr Leu Gly Val
Ser Pro Thr Val Gln Gly 2900 2905 2910Leu Val Thr Ser Leu Val Thr
Ser Ser Gly Ser Glu Thr Ser Ala Phe 2915 2920 2925Ser Asn Leu Thr
Val Ala Ser Ser Gln Pro Glu Thr Ile Asp Ser Trp 2930 2935 2940Val
Ala His Pro Gly Thr Glu Ala Ser Ser Val Val Pro Thr Leu Thr2945
2950 2955 2960Val Ser Thr Gly Glu Pro Phe Thr Asn Ile Ser Leu Val
Thr His Pro 2965 2970 2975Ala Glu Ser Ser Ser Thr Leu Pro Arg Thr
Thr Ser Arg Phe Ser His 2980 2985 2990Ser Glu Leu Asp Thr Met Pro
Ser Thr Val Thr Ser Pro Glu Ala Glu 2995 3000 3005Ser Ser Ser Ala
Ile Ser Thr Thr Ile Ser Pro Gly Ile Pro Gly Val 3010 3015 3020Leu
Thr Ser Leu Val Thr Ser Ser Gly Arg Asp Ile Ser Ala Thr Phe3025
3030 3035 3040Pro Thr Val Pro Glu Ser Pro His Glu Ser Glu Ala Thr
Ala Ser Trp 3045 3050 3055Val Thr His Pro Ala Val Thr Ser Thr Thr
Val Pro Arg Thr Thr Pro 3060 3065 3070Asn Tyr Ser His Ser Glu Pro
Asp Thr Thr Pro Ser Ile Ala Thr Ser 3075 3080 3085Pro Gly Ala Glu
Ala Thr Ser Asp Phe Pro Thr Ile Thr Val Ser Pro 3090 3095 3100Asp
Val Pro Asp Met Val Thr Ser Gln Val Thr Ser Ser Gly Thr Asp3105
3110 3115 3120Thr Ser Ile Thr Ile Pro Thr Leu Thr Leu Ser Ser Gly
Glu Pro Glu 3125 3130 3135Thr Thr Thr Ser Phe Ile Thr Tyr Ser Glu
Thr His Thr Ser Ser Ala 3140 3145 3150Ile Pro Thr Leu Pro Val Ser
Pro Asp Ala Ser Lys Met Leu Thr Ser 3155 3160 3165Leu Val Ile Ser
Ser Gly Thr Asp Ser Thr Thr Thr Phe Pro Thr Leu 3170 3175 3180Thr
Glu Thr Pro Tyr Glu Pro Glu Thr Thr Ala Ile Gln Leu Ile His3185
3190 3195 3200Pro Ala Glu Thr Asn Thr Met Val Pro Arg Thr Thr Pro
Lys Phe Ser 3205 3210 3215His Ser Lys Ser Asp Thr Thr Leu Pro Val
Ala Ile Thr Ser Pro Gly 3220 3225 3230Pro Glu Ala Ser Ser Ala Val
Ser Thr Thr Thr Ile Ser Pro Asp Met 3235 3240 3245Ser Asp Leu Val
Thr Ser Leu Val Pro Ser Ser Gly Thr Asp Thr Ser 3250 3255 3260Thr
Thr Phe Pro Thr Leu Ser Glu Thr Pro Tyr Glu Pro Glu Thr Thr3265
3270 3275 3280Ala Thr Trp Leu Thr His Pro Ala Glu Thr Ser Thr Thr
Val Ser Gly 3285 3290 3295Thr Ile Pro Asn Phe Ser His Arg Gly Ser
Asp Thr Ala Pro Ser Met 3300 3305 3310Val Thr Ser Pro Gly Val Asp
Thr Arg Ser Gly Val Pro Thr Thr Thr 3315 3320 3325Ile Pro Pro Ser
Ile Pro Gly Val Val Thr Ser Gln Val Thr Ser Ser 3330 3335 3340Ala
Thr Asp Thr Ser Thr Ala Ile Pro Thr Leu Thr Pro Ser Pro Gly3345
3350 3355 3360Glu Pro Glu Thr Thr Ala Ser Ser Ala Thr His Pro Gly
Thr Gln Thr 3365 3370 3375Gly Phe Thr Val Pro Ile Arg Thr Val Pro
Ser Ser Glu Pro Asp Thr 3380 3385 3390Met Ala Ser Trp Val Thr His
Pro Pro Gln Thr Ser Thr Pro Val Ser 3395 3400 3405Arg Thr Thr Ser
Ser Phe Ser His Ser Ser Pro Asp Ala Thr Pro Val 3410 3415 3420Met
Ala Thr Ser Pro Arg Thr Glu Ala Ser Ser Ala Val Leu Thr Thr3425
3430 3435 3440Ile Ser Pro Gly Ala Pro Glu Met Val Thr Ser Gln Ile
Thr Ser Ser 3445 3450 3455Gly Ala Ala Thr Ser Thr Thr Val Pro Thr
Leu Thr His Ser Pro Gly 3460 3465 3470Met Pro Glu Thr Thr Ala Leu
Leu Ser Thr His Pro Arg Thr Glu Thr 3475 3480 3485Ser Lys Thr Phe
Pro Ala Ser Thr Val Phe Pro Gln Val Ser Glu Thr 3490 3495 3500Thr
Ala Ser Leu Thr Ile Arg Pro Gly Ala Glu Thr Ser Thr Ala Leu3505
3510 3515 3520Pro Thr Gln Thr Thr Ser Ser Leu Phe Thr Leu Leu Val
Thr Gly Thr 3525 3530 3535Ser Arg Val Asp Leu Ser Pro Thr Ala Ser
Pro Gly Val Ser Ala Lys 3540 3545 3550Thr Ala Pro Leu Ser Thr His
Pro Gly Thr Glu Thr Ser Thr Met Ile 3555 3560 3565Pro Thr Ser Thr
Leu Ser Leu
Gly Leu Leu Glu Thr Thr Gly Leu Leu 3570 3575 3580Ala Thr Ser Ser
Ser Ala Glu Thr Ser Thr Ser Thr Leu Thr Leu Thr3585 3590 3595
3600Val Ser Pro Ala Val Ser Gly Leu Ser Ser Ala Ser Ile Thr Thr Asp
3605 3610 3615Lys Pro Gln Thr Val Thr Ser Trp Asn Thr Glu Thr Ser
Pro Ser Val 3620 3625 3630Thr Ser Val Gly Pro Pro Glu Phe Ser Arg
Thr Val Thr Gly Thr Thr 3635 3640 3645Met Thr Leu Ile Pro Ser Glu
Met Pro Thr Pro Pro Lys Thr Ser His 3650 3655 3660Gly Glu Gly Val
Ser Pro Thr Thr Ile Leu Arg Thr Thr Met Val Glu3665 3670 3675
3680Ala Thr Asn Leu Ala Thr Thr Gly Ser Ser Pro Thr Val Ala Lys Thr
3685 3690 3695Thr Thr Thr Phe Asn Thr Leu Ala Gly Ser Leu Phe Thr
Pro Leu Thr 3700 3705 3710Thr Pro Gly Met Ser Thr Leu Ala Ser Glu
Ser Val Thr Ser Arg Thr 3715 3720 3725Ser Tyr Asn His Arg Ser Trp
Ile Ser Thr Thr Ser Ser Tyr Asn Arg 3730 3735 3740Arg Tyr Trp Thr
Pro Ala Thr Ser Thr Pro Val Thr Ser Thr Phe Ser3745 3750 3755
3760Pro Gly Ile Ser Thr Ser Ser Ile Pro Ser Ser Thr Ala Ala Thr Val
3765 3770 3775Pro Phe Met Val Pro Phe Thr Leu Asn Phe Thr Ile Thr
Asn Leu Gln 3780 3785 3790Tyr Glu Glu Asp Met Arg His Pro Gly Ser
Arg Lys Phe Asn Ala Thr 3795 3800 3805Glu Arg Glu Leu Gln Gly Leu
Leu Lys Pro Leu Phe Arg Asn Ser Ser 3810 3815 3820Leu Glu Tyr Leu
Tyr Ser Gly Cys Arg Leu Ala Ser Leu Arg Pro Glu3825 3830 3835
3840Lys Asp Ser Ser Ala Thr Ala Val Asp Ala Ile Cys Thr His Arg Pro
3845 3850 3855Asp Pro Glu Asp Leu Gly Leu Asp Arg Glu Arg Leu Tyr
Trp Glu Leu 3860 3865 3870Ser Asn Leu Thr Asn Gly Ile Gln Glu Leu
Gly Pro Tyr Thr Leu Asp 3875 3880 3885Arg Asn Ser Leu Tyr Val Asn
Gly Phe Thr His Arg Ser Ser Met Pro 3890 3895 3900Thr Thr Ser Thr
Pro Gly Thr Ser Thr Val Asp Val Gly Thr Ser Gly3905 3910 3915
3920Thr Pro Ser Ser Ser Pro Ser Pro Thr Thr Ala Gly Pro Leu Leu Met
3925 3930 3935Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr
Glu Glu Asp 3940 3945 3950Met Arg Arg Thr Gly Ser Arg Lys Phe Asn
Thr Met Glu Ser Val Leu 3955 3960 3965Gln Gly Leu Leu Lys Pro Leu
Phe Lys Asn Thr Ser Val Gly Pro Leu 3970 3975 3980Tyr Ser Gly Cys
Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Ala3985 3990 3995
4000Ala Thr Gly Val Asp Ala Ile Cys Thr His Arg Leu Asp Pro Lys Ser
4005 4010 4015Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp Glu Leu Ser
Lys Leu Thr 4020 4025 4030Asn Asp Ile Glu Glu Leu Gly Pro Tyr Thr
Leu Asp Arg Asn Ser Leu 4035 4040 4045Tyr Val Asn Gly Phe Thr His
Gln Ser Ser Val Ser Thr Thr Ser Thr 4050 4055 4060Pro Gly Thr Ser
Thr Val Asp Leu Arg Thr Ser Gly Thr Pro Ser Ser4065 4070 4075
4080Leu Ser Ser Pro Thr Ile Met Ala Ala Gly Pro Leu Leu Val Pro Phe
4085 4090 4095Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Gly Glu
Asp Met Gly 4100 4105 4110His Pro Gly Ser Arg Lys Phe Asn Thr Thr
Glu Arg Val Leu Gln Gly 4115 4120 4125Leu Leu Gly Pro Ile Phe Lys
Asn Thr Ser Val Gly Pro Leu Tyr Ser 4130 4135 4140Gly Cys Arg Leu
Thr Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr4145 4150 4155
4160Gly Val Asp Ala Ile Cys Ile His His Leu Asp Pro Lys Ser Pro Gly
4165 4170 4175Leu Asn Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu
Thr Asn Gly 4180 4185 4190Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp
Arg Asn Ser Leu Tyr Val 4195 4200 4205Asn Gly Phe Thr His Arg Thr
Ser Val Pro Thr Thr Ser Thr Pro Gly 4210 4215 4220Thr Ser Thr Val
Asp Leu Gly Thr Ser Gly Thr Pro Phe Ser Leu Pro4225 4230 4235
4240Ser Pro Ala Thr Ala Gly Pro Leu Leu Val Leu Phe Thr Leu Asn Phe
4245 4250 4255Thr Ile Thr Asn Leu Lys Tyr Glu Glu Asp Met His Arg
Pro Gly Ser 4260 4265 4270Arg Lys Phe Asn Thr Thr Glu Arg Val Leu
Gln Thr Leu Val Gly Pro 4275 4280 4285Met Phe Lys Asn Thr Ser Val
Gly Leu Leu Tyr Ser Gly Cys Arg Leu 4290 4295 4300Thr Leu Leu Arg
Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala4305 4310 4315
4320Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Val Asp Arg Glu
4325 4330 4335Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile
Lys Glu Leu 4340 4345 4350Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu
Tyr Val Asn Gly Phe Thr 4355 4360 4365His Trp Ile Pro Val Pro Thr
Ser Ser Thr Pro Gly Thr Ser Thr Val 4370 4375 4380Asp Leu Gly Ser
Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala4385 4390 4395
4400Thr Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr
4405 4410 4415Asn Leu Lys Tyr Glu Glu Asp Met His Cys Pro Gly Ser
Arg Lys Phe 4420 4425 4430Asn Thr Thr Glu Arg Val Leu Gln Ser Leu
Leu Gly Pro Met Phe Lys 4435 4440 4445Asn Thr Ser Val Gly Pro Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu 4450 4455 4460Arg Ser Glu Lys
Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr4465 4470 4475
4480His Arg Leu Asp Pro Lys Ser Pro Gly Val Asp Arg Glu Gln Leu Tyr
4485 4490 4495Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu
Gly Pro Tyr 4500 4505 4510Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn
Gly Phe Thr His Gln Thr 4515 4520 4525Ser Ala Pro Asn Thr Ser Thr
Pro Gly Thr Ser Thr Val Asp Leu Gly 4530 4535 4540Thr Ser Gly Thr
Pro Ser Ser Leu Pro Ser Pro Thr Ser Ala Gly Pro4545 4550 4555
4560Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr
4565 4570 4575Glu Glu Asp Met His His Pro Gly Ser Arg Lys Phe Asn
Thr Thr Glu 4580 4585 4590Arg Val Leu Gln Gly Leu Leu Gly Pro Met
Phe Lys Asn Thr Ser Val 4595 4600 4605Gly Leu Leu Tyr Ser Gly Cys
Arg Leu Thr Leu Leu Arg Pro Glu Lys 4610 4615 4620Asn Gly Ala Ala
Thr Gly Met Asp Ala Ile Cys Ser His Arg Leu Asp4625 4630 4635
4640Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp Glu Leu Ser
4645 4650 4655Gln Leu Thr His Gly Ile Lys Glu Leu Gly Pro Tyr Thr
Leu Asp Arg 4660 4665 4670Asn Ser Leu Tyr Val Asn Gly Phe Thr His
Arg Ser Ser Val Ala Pro 4675 4680 4685Thr Ser Thr Pro Gly Thr Ser
Thr Val Asp Leu Gly Thr Ser Gly Thr 4690 4695 4700Pro Ser Ser Leu
Pro Ser Pro Thr Thr Ala Val Pro Leu Leu Val Pro4705 4710 4715
4720Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Gly Glu Asp Met
4725 4730 4735Arg His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg
Val Leu Gln 4740 4745 4750Gly Leu Leu Gly Pro Leu Phe Lys Asn Ser
Ser Val Gly Pro Leu Tyr 4755 4760 4765Ser Gly Cys Arg Leu Ile Ser
Leu Arg Ser Glu Lys Asp Gly Ala Ala 4770 4775 4780Thr Gly Val Asp
Ala Ile Cys Thr His His Leu Asn Pro Gln Ser Pro4785 4790 4795
4800Gly Leu Asp Arg Glu Gln Leu Tyr Trp Gln Leu Ser Gln Met Thr Asn
4805 4810 4815Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asn
Ser Leu Tyr 4820 4825 4830Val Asn Gly Phe Thr His Arg Ser Ser Gly
Leu Thr Thr Ser Thr Pro 4835 4840 4845Trp Thr Ser Thr Val Asp Leu
Gly Thr Ser Gly Thr Pro Ser Pro Val 4850 4855 4860Pro Ser Pro Thr
Thr Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn4865 4870 4875
4880Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His Arg Pro Gly
4885 4890 4895Ser Arg Lys Phe Asn Ala Thr Glu Arg Val Leu Gln Gly
Leu Leu Ser 4900 4905 4910Pro Ile Phe Lys Asn Ser Ser Val Gly Pro
Leu Tyr Ser Gly Cys Arg 4915 4920 4925Leu Thr Ser Leu Arg Pro Glu
Lys Asp Gly Ala Ala Thr Gly Met Asp 4930 4935 4940Ala Val Cys Leu
Tyr His Pro Asn Pro Lys Arg Pro Gly Leu Asp Arg4945 4950 4955
4960Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Asn Ile Thr Glu
4965 4970 4975Leu Gly Pro Tyr Ser Leu Asp Arg Asp Ser Leu Tyr Val
Asn Gly Phe 4980 4985 4990Thr His Gln Asn Ser Val Pro Thr Thr Ser
Thr Pro Gly Thr Ser Thr 4995 5000 5005Val Tyr Trp Ala Thr Thr Gly
Thr Pro Ser Ser Phe Pro Gly His Thr 5010 5015 5020Glu Pro Gly Pro
Leu Leu Ile Pro Phe Thr Phe Asn Phe Thr Ile Thr5025 5030 5035
5040Asn Leu His Tyr Glu Glu Asn Met Gln His Pro Gly Ser Arg Lys Phe
5045 5050 5055Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Lys Pro
Leu Phe Lys 5060 5065 5070Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly
Cys Arg Leu Thr Leu Leu 5075 5080 5085Arg Pro Glu Lys Gln Glu Ala
Ala Thr Gly Val Asp Thr Ile Cys Thr 5090 5095 5100His Arg Val Asp
Pro Ile Gly Pro Gly Leu Asp Arg Glu Arg Leu Tyr5105 5110 5115
5120Trp Glu Leu Ser Gln Leu Thr Asn Ser Ile Thr Glu Leu Gly Pro Tyr
5125 5130 5135Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Asn
Pro Trp Ser 5140 5145 5150Ser Val Pro Thr Thr Ser Thr Pro Gly Thr
Ser Thr Val His Leu Ala 5155 5160 5165Thr Ser Gly Thr Pro Ser Ser
Leu Pro Gly His Thr Ala Pro Val Pro 5170 5175 5180Leu Leu Ile Pro
Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu His Tyr5185 5190 5195
5200Glu Glu Asn Met Gln His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu
5205 5210 5215Arg Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Ser
Thr Ser Val 5220 5225 5230Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr
Leu Leu Arg Pro Glu Lys 5235 5240 5245His Gly Ala Ala Thr Gly Val
Asp Ala Ile Cys Thr Leu Arg Leu Asp 5250 5255 5260Pro Thr Gly Pro
Gly Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser5265 5270 5275
5280Gln Leu Thr Asn Ser Val Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg
5285 5290 5295Asp Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser
Val Pro Thr 5300 5305 5310Thr Ser Ile Pro Gly Thr Ser Ala Val His
Leu Glu Thr Ser Gly Thr 5315 5320 5325Pro Ala Ser Leu Pro Gly His
Thr Ala Pro Gly Pro Leu Leu Val Pro 5330 5335 5340Phe Thr Leu Asn
Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met5345 5350 5355
5360Arg His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln
5365 5370 5375Gly Leu Leu Lys Pro Leu Phe Lys Ser Thr Ser Val Gly
Pro Leu Tyr 5380 5385 5390Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro
Glu Lys Arg Gly Ala Ala 5395 5400 5405Thr Gly Val Asp Thr Ile Cys
Thr His Arg Leu Asp Pro Leu Asn Pro 5410 5415 5420Gly Leu Asp Arg
Glu Gln Leu Tyr Trp Glu Leu Ser Lys Leu Thr Arg5425 5430 5435
5440Gly Ile Ile Glu Leu Gly Pro Tyr Leu Leu Asp Arg Gly Ser Leu Tyr
5445 5450 5455Val Asn Gly Phe Thr His Arg Asn Phe Val Pro Ile Thr
Ser Thr Pro 5460 5465 5470Gly Thr Ser Thr Val His Leu Gly Thr Ser
Glu Thr Pro Ser Ser Leu 5475 5480 5485Pro Arg Pro Ile Val Pro Gly
Pro Leu Leu Val Pro Phe Thr Leu Asn 5490 5495 5500Phe Thr Ile Thr
Asn Leu Gln Tyr Glu Glu Ala Met Arg His Pro Gly5505 5510 5515
5520Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg
5525 5530 5535Pro Leu Phe Lys Asn Thr Ser Ile Gly Pro Leu Tyr Ser
Ser Cys Arg 5540 5545 5550Leu Thr Leu Leu Arg Pro Glu Lys Asp Lys
Ala Ala Thr Arg Val Asp 5555 5560 5565Ala Ile Cys Thr His His Pro
Asp Pro Gln Ser Pro Gly Leu Asn Arg 5570 5575 5580Glu Gln Leu Tyr
Trp Glu Leu Ser Gln Leu Thr His Gly Ile Thr Glu5585 5590 5595
5600Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val Asp Gly Phe
5605 5610 5615Thr His Trp Ser Pro Ile Pro Thr Thr Ser Thr Pro Gly
Thr Ser Ile 5620 5625 5630Val Asn Leu Gly Thr Ser Gly Ile Pro Pro
Ser Leu Pro Glu Thr Thr 5635 5640 5645Ala Thr Gly Pro Leu Leu Val
Pro Phe Thr Leu Asn Phe Thr Ile Thr 5650 5655 5660Asn Leu Gln Tyr
Glu Glu Asn Met Gly His Pro Gly Ser Arg Lys Phe5665 5670 5675
5680Asn Ile Thr Glu Ser Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys
5685 5690 5695Ser Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu
Thr Leu Leu 5700 5705 5710Arg Pro Glu Lys Asp Gly Val Ala Thr Arg
Val Asp Ala Ile Cys Thr 5715 5720 5725His Arg Pro Asp Pro Lys Ile
Pro Gly Leu Asp Arg Gln Gln Leu Tyr 5730 5735 5740Trp Glu Leu Ser
Gln Leu Thr His Ser Ile Thr Glu Leu Gly Pro Tyr5745 5750 5755
5760Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr Gln Arg Ser
5765 5770 5775Ser Val Pro Thr Thr Ser Thr Pro Gly Thr Phe Thr Val
Gln Pro Glu 5780 5785 5790Thr Ser Glu Thr Pro Ser Ser Leu Pro Gly
Pro Thr Ala Thr Gly Pro 5795 5800 5805Val Leu Leu Pro Phe Thr Leu
Asn Phe Thr Ile Ile Asn Leu Gln Tyr 5810 5815 5820Glu Glu Asp Met
His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu5825 5830 5835
5840Arg Val Leu Gln Gly Leu Leu Met Pro Leu Phe Lys Asn Thr Ser Val
5845 5850 5855Ser Ser Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg
Pro Glu Lys 5860 5865 5870Asp Gly Ala Ala Thr Arg Val Asp Ala Val
Cys Thr His Arg Pro Asp 5875 5880 5885Pro Lys Ser Pro Gly Leu Asp
Arg Glu Arg Leu Tyr Trp Lys Leu Ser 5890 5895 5900Gln Leu Thr His
Gly Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg5905 5910 5915
5920His Ser Leu Tyr Val Asn Gly Phe Thr His Gln Ser Ser Met Thr Thr
5925 5930 5935Thr Arg Thr Pro Asp Thr Ser Thr Met His Leu Ala Thr
Ser Arg Thr 5940 5945 5950Pro Ala Ser Leu Ser Gly Pro Thr Thr Ala
Ser Pro Leu Leu Val Leu 5955 5960 5965Phe Thr Ile Asn Phe Thr Ile
Thr Asn Leu Arg Tyr Glu Glu Asn Met 5970 5975 5980His His Pro Gly
Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln5985 5990 5995
6000Gly Leu Leu Arg Pro Val Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr
6005 6010 6015Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Lys Lys Asp
Gly Ala Ala 6020 6025
6030Thr Lys Val Asp Ala Ile Cys Thr Tyr Arg Pro Asp Pro Lys Ser Pro
6035 6040 6045Gly Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln
Leu Thr His 6050 6055 6060Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu
Asp Arg Asp Ser Leu Tyr6065 6070 6075 6080Val Asn Gly Phe Thr Gln
Arg Ser Ser Val Pro Thr Thr Ser Ile Pro 6085 6090 6095Gly Thr Pro
Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Val Ser Lys 6100 6105
6110Pro Gly Pro Ser Ala Ala Ser Pro Leu Leu Val Leu Phe Thr Leu Asn
6115 6120 6125Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met Gln
His Pro Gly 6130 6135 6140Ser Arg Lys Phe Asn Thr Thr Glu Arg Val
Leu Gln Gly Leu Leu Arg6145 6150 6155 6160Ser Leu Phe Lys Ser Thr
Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg 6165 6170 6175Leu Thr Leu
Leu Arg Pro Glu Lys Asp Gly Thr Ala Thr Gly Val Asp 6180 6185
6190Ala Ile Cys Thr His His Pro Asp Pro Lys Ser Pro Arg Leu Asp Arg
6195 6200 6205Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Asn
Ile Thr Glu 6210 6215 6220Leu Gly Pro Tyr Ala Leu Asp Asn Asp Ser
Leu Phe Val Asn Gly Phe6225 6230 6235 6240Thr His Arg Ser Ser Val
Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr 6245 6250 6255Val Tyr Leu
Gly Ala Ser Lys Thr Pro Ala Ser Ile Phe Gly Pro Ser 6260 6265
6270Ala Ala Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr
6275 6280 6285Asn Leu Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg
Lys Phe Asn 6290 6295 6300Thr Thr Glu Arg Val Leu Gln Gly Leu Leu
Arg Pro Leu Phe Lys Asn6305 6310 6315 6320Thr Ser Val Gly Pro Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg 6325 6330 6335Pro Glu Lys
Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr His 6340 6345
6350Arg Pro Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu Tyr Leu
6355 6360 6365Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly
Pro Tyr Thr 6370 6375 6380Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly
Phe Thr His Arg Ser Ser6385 6390 6395 6400Val Pro Thr Thr Ser Thr
Gly Val Val Ser Glu Glu Pro Phe Thr Leu 6405 6410 6415Asn Phe Thr
Ile Asn Asn Leu Arg Tyr Met Ala Asp Met Gly Gln Pro 6420 6425
6430Gly Ser Leu Lys Phe Asn Ile Thr Asp Asn Val Met Gln His Leu Leu
6435 6440 6445Ser Pro Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr
Thr Gly Cys 6450 6455 6460Arg Val Ile Ala Leu Arg Ser Val Lys Asn
Gly Ala Glu Thr Arg Val6465 6470 6475 6480Asp Leu Leu Cys Thr Tyr
Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro 6485 6490 6495Ile Lys Gln
Val Phe His Glu Leu Ser Gln Gln Thr His Gly Ile Thr 6500 6505
6510Arg Leu Gly Pro Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu Asn Gly
6515 6520 6525Tyr Asn Glu Pro Gly Pro Asp Glu Pro Pro Thr Thr Pro
Lys Pro Ala 6530 6535 6540Thr Thr Phe Leu Pro Pro Leu Ser Glu Ala
Thr Thr Ala Met Gly Tyr6545 6550 6555 6560His Leu Lys Thr Leu Thr
Leu Asn Phe Thr Ile Ser Asn Leu Gln Tyr 6565 6570 6575Ser Pro Asp
Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu Gly 6580 6585
6590Val Leu Gln His Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser Met Gly
6595 6600 6605Pro Phe Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro
Glu Lys Asp 6610 6615 6620Gly Ala Ala Thr Gly Val Asp Thr Thr Cys
Thr Tyr His Pro Asp Pro6625 6630 6635 6640Val Gly Pro Gly Leu Asp
Ile Gln Gln Leu Tyr Trp Glu Leu Ser Gln 6645 6650 6655Leu Thr His
Gly Val Thr Gln Leu Gly Phe Tyr Val Leu Asp Arg Asp 6660 6665
6670Ser Leu Phe Ile Asn Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly
6675 6680 6685Glu Tyr Gln Ile Asn Phe His Ile Val Asn Trp Asn Leu
Ser Asn Pro 6690 6695 6700Asp Pro Thr Ser Ser Glu Tyr Ile Thr Leu
Leu Arg Asp Ile Gln Asp6705 6710 6715 6720Lys Val Thr Thr Leu Tyr
Lys Gly Ser Gln Leu His Asp Thr Phe Arg 6725 6730 6735Phe Cys Leu
Val Thr Asn Leu Thr Met Asp Ser Val Leu Val Thr Val 6740 6745
6750Lys Ala Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln Val
6755 6760 6765Phe Leu Asp Lys Thr Leu Asn Ala Ser Phe His Trp Leu
Gly Ser Thr 6770 6775 6780Tyr Gln Leu Val Asp Ile His Val Thr Glu
Met Glu Ser Ser Val Tyr6785 6790 6795 6800Gln Pro Thr Ser Ser Ser
Ser Thr Gln His Phe Tyr Leu Asn Phe Thr 6805 6810 6815Ile Thr Asn
Leu Pro Tyr Ser Gln Asp Lys Ala Gln Pro Gly Thr Thr 6820 6825
6830Asn Tyr Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu Asn Gln Leu
6835 6840 6845Phe Arg Asn Ser Ser Ile Lys Ser Tyr Phe Ser Asp Cys
Gln Val Ser 6850 6855 6860Thr Phe Arg Ser Val Pro Asn Arg His His
Thr Gly Val Asp Ser Leu6865 6870 6875 6880Cys Asn Phe Ser Pro Leu
Ala Arg Arg Val Asp Arg Val Ala Ile Tyr 6885 6890 6895Glu Glu Phe
Leu Arg Met Thr Arg Asn Gly Thr Gln Leu Gln Asn Phe 6900 6905
6910Thr Leu Asp Arg Ser Ser Val Leu Val Asp Gly Tyr Ser Pro Asn Arg
6915 6920 6925Asn Glu Pro Leu Thr Gly Asn Ser Asp Leu Pro Phe Trp
Ala Val Ile 6930 6935 6940Leu Ile Gly Leu Ala Gly Leu Leu Gly Leu
Ile Thr Cys Leu Ile Cys6945 6950 6955 6960Gly Val Leu Val Thr Thr
Arg Arg Arg Lys Lys Glu Gly Glu Tyr Asn 6965 6970 6975Val Gln Gln
Gln Cys Pro Gly Tyr Tyr Gln Ser His Leu Asp Leu Glu 6980 6985
6990Asp Leu Gln 69955622PRTHomo sapiensMSLN 5Met Ala Leu Pro Thr
Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro1 5 10 15Ala Leu Gly Ser
Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30Pro Ser Arg
Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu 35 40 45Asp Gly
Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg 50 55 60Gln
Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu65 70 75
80Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu
85 90 95Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro
Pro 100 105 110Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe
Leu Asn Pro 115 120 125Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg
Phe Phe Ser Arg Ile 130 135 140Thr Lys Ala Asn Val Asp Leu Leu Pro
Arg Gly Ala Pro Glu Arg Gln145 150 155 160Arg Leu Leu Pro Ala Ala
Leu Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170 175Leu Ser Glu Ala
Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185 190Pro Gly
Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu 195 200
205Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg
210 215 220Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser
Thr Trp225 230 235 240Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu
Leu Pro Val Leu Gly 245 250 255Gln Pro Ile Ile Arg Ser Ile Pro Gln
Gly Ile Val Ala Ala Trp Arg 260 265 270Gln Arg Ser Ser Arg Asp Pro
Ser Trp Arg Gln Pro Glu Arg Thr Ile 275 280 285Leu Arg Pro Arg Phe
Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295 300Gly Lys Lys
Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys305 310 315
320Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met
325 330 335Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp
Val Leu 340 345 350Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr
Pro Glu Ser Val 355 360 365Ile Gln His Leu Gly Tyr Leu Phe Leu Lys
Met Ser Pro Glu Asp Ile 370 375 380Arg Lys Trp Asn Val Thr Ser Leu
Glu Thr Leu Lys Ala Leu Leu Glu385 390 395 400Val Asn Lys Gly His
Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp 405 410 415Arg Phe Val
Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr 420 425 430Leu
Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu 435 440
445Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp
450 455 460Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro
Lys Ala465 470 475 480Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu
Tyr Phe Val Lys Ile 485 490 495Gln Ser Phe Leu Gly Gly Ala Pro Thr
Glu Asp Leu Lys Ala Leu Ser 500 505 510Gln Gln Asn Val Ser Met Asp
Leu Ala Thr Phe Met Lys Leu Arg Thr 515 520 525Asp Ala Val Leu Pro
Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly 530 535 540Pro His Val
Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg545 550 555
560Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu
565 570 575Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp
Leu Ser 580 585 590Met Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu
Gly Pro Gly Pro 595 600 605Val Leu Thr Val Leu Ala Leu Leu Leu Ala
Ser Thr Leu Ala 610 615 6206690PRTHomo sapiensSLC34A2 6Met Ala Pro
Trp Pro Glu Leu Gly Asp Ala Gln Pro Asn Pro Asp Lys1 5 10 15Tyr Leu
Glu Gly Ala Ala Gly Gln Gln Pro Thr Ala Pro Asp Lys Ser 20 25 30Lys
Glu Thr Asn Lys Thr Asp Asn Thr Glu Ala Pro Val Thr Lys Ile 35 40
45Glu Leu Leu Pro Ser Tyr Ser Thr Ala Thr Leu Ile Asp Glu Pro Thr
50 55 60Glu Val Asp Asp Pro Trp Asn Leu Pro Thr Leu Gln Asp Ser Gly
Ile65 70 75 80Lys Trp Ser Glu Arg Asp Thr Lys Gly Lys Ile Leu Cys
Phe Phe Gln 85 90 95Gly Ile Gly Arg Leu Ile Leu Leu Leu Gly Phe Leu
Tyr Phe Phe Val 100 105 110Cys Ser Leu Asp Ile Leu Ser Ser Ala Phe
Gln Leu Val Gly Gly Lys 115 120 125Met Ala Gly Gln Phe Phe Ser Asn
Ser Ser Ile Met Ser Asn Pro Leu 130 135 140Leu Gly Leu Val Ile Gly
Val Leu Val Thr Val Leu Val Gln Ser Ser145 150 155 160Ser Thr Ser
Thr Ser Ile Val Val Ser Met Val Ser Ser Ser Leu Leu 165 170 175Thr
Val Arg Ala Ala Ile Pro Ile Ile Met Gly Ala Asn Ile Gly Thr 180 185
190Ser Ile Thr Asn Thr Ile Val Ala Leu Met Gln Val Gly Asp Arg Ser
195 200 205Glu Phe Arg Arg Ala Phe Ala Gly Ala Thr Val His Asp Phe
Phe Asn 210 215 220Trp Leu Ser Val Leu Val Leu Leu Pro Val Glu Val
Ala Thr His Tyr225 230 235 240Leu Glu Ile Ile Thr Gln Leu Ile Val
Glu Ser Phe His Phe Lys Asn 245 250 255Gly Glu Asp Ala Pro Asp Leu
Leu Lys Val Ile Thr Lys Pro Phe Thr 260 265 270Lys Leu Ile Val Gln
Leu Asp Lys Lys Val Ile Ser Gln Ile Ala Met 275 280 285Asn Asp Glu
Lys Ala Lys Asn Lys Ser Leu Val Lys Ile Trp Cys Lys 290 295 300Thr
Phe Thr Asn Lys Thr Gln Ile Asn Val Thr Val Pro Ser Thr Ala305 310
315 320Asn Cys Thr Ser Pro Ser Leu Cys Trp Thr Asp Gly Ile Gln Asn
Trp 325 330 335Thr Met Lys Asn Val Thr Tyr Lys Glu Asn Ile Ala Lys
Cys Gln His 340 345 350Ile Phe Val Asn Phe His Leu Pro Asp Leu Ala
Val Gly Thr Ile Leu 355 360 365Leu Ile Leu Ser Leu Leu Val Leu Cys
Gly Cys Leu Ile Met Ile Val 370 375 380Lys Ile Leu Gly Ser Val Leu
Lys Gly Gln Val Ala Thr Val Ile Lys385 390 395 400Lys Thr Ile Asn
Thr Asp Phe Pro Phe Pro Phe Ala Trp Leu Thr Gly 405 410 415Tyr Leu
Ala Ile Leu Val Gly Ala Gly Met Thr Phe Ile Val Gln Ser 420 425
430Ser Ser Val Phe Thr Ser Ala Leu Thr Pro Leu Ile Gly Ile Gly Val
435 440 445Ile Thr Ile Glu Arg Ala Tyr Pro Leu Thr Leu Gly Ser Asn
Ile Gly 450 455 460Thr Thr Thr Thr Ala Ile Leu Ala Ala Leu Ala Ser
Pro Gly Asn Ala465 470 475 480Leu Arg Ser Ser Leu Gln Ile Ala Leu
Cys His Phe Phe Phe Asn Ile 485 490 495Ser Gly Ile Leu Leu Trp Tyr
Pro Ile Pro Phe Thr Arg Leu Pro Ile 500 505 510Arg Met Ala Lys Gly
Leu Gly Asn Ile Ser Ala Lys Tyr Arg Trp Phe 515 520 525Ala Val Phe
Tyr Leu Ile Ile Phe Phe Phe Leu Ile Pro Leu Thr Val 530 535 540Phe
Gly Leu Ser Leu Ala Gly Trp Arg Val Leu Val Gly Val Gly Val545 550
555 560Pro Val Val Phe Ile Ile Ile Leu Val Leu Cys Leu Arg Leu Leu
Gln 565 570 575Ser Arg Cys Pro Arg Val Leu Pro Lys Lys Leu Gln Asn
Trp Asn Phe 580 585 590Leu Pro Leu Trp Met Arg Ser Leu Lys Pro Trp
Asp Ala Val Val Ser 595 600 605Lys Phe Thr Gly Cys Phe Gln Met Arg
Cys Cys Tyr Cys Cys Arg Val 610 615 620Cys Cys Arg Ala Cys Cys Leu
Leu Cys Gly Cys Pro Lys Cys Cys Arg625 630 635 640Cys Ser Lys Cys
Cys Glu Asp Leu Glu Glu Ala Gln Glu Gly Gln Asp 645 650 655Val Pro
Val Lys Ala Pro Glu Thr Phe Asp Asn Ile Thr Ile Ser Arg 660 665
670Glu Ala Gln Gly Glu Val Pro Ala Ser Asp Ser Lys Thr Glu Cys Thr
675 680 685Ala Leu 69071093PRTHomo sapiensKIAA1445 7Met Val Leu Ala
Gly Pro Leu Ala Val Ser Leu Leu Leu Pro Ser Leu1 5 10 15Thr Leu Leu
Val Ser His Leu Ser Ser Ser Gln Asp Val Ser Ser Glu 20 25 30Pro Ser
Ser Glu Gln Gln Leu Cys Ala Leu Ser Lys His Pro Thr Val 35 40 45Ala
Phe Glu Asp Leu Gln Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly 50 55
60Ala Arg Asp Phe Ser Gln Leu Ala Leu Asp Pro Ser Gly Asn Gln Leu65
70 75 80Ile Val Gly Ala Arg Asn Tyr Leu Phe Arg Leu Ser Leu Ala Asn
Val 85 90 95Ser Leu Leu Gln Ala Thr Glu Trp Ala Ser Ser Glu Asp Thr
Arg Arg 100 105 110Ser Cys Gln Ser Lys Gly Lys Thr Glu Glu Glu Cys
Gln Asn Tyr Val 115 120 125Arg Val Leu Ile Val Ala Gly Arg Lys Val
Phe Met Cys Gly Thr Asn 130 135 140Ala Phe Ser Pro Met Cys Thr Ser
Arg Gln Val Gly Asn Leu Ser Arg145 150 155 160Thr Thr Glu Lys Ile
Asn Gly Val Ala Arg Cys Pro Tyr Asp Pro Arg 165 170 175His Asn Ser
Thr Ala Val Ile
Ser Ser Gln Gly Glu Leu Tyr Ala Ala 180 185 190Thr Val Ile Asp Phe
Ser Gly Arg Asp Pro Ala Ile Tyr Arg Ser Leu 195 200 205Gly Ser Gly
Pro Pro Leu Arg Thr Ala Gln Tyr Asn Ser Lys Trp Leu 210 215 220Asn
Glu Pro Asn Phe Val Ala Ala Tyr Asp Ile Gly Leu Phe Ala Tyr225 230
235 240Phe Phe Leu Arg Glu Asn Ala Val Glu His Asp Cys Gly Arg Thr
Val 245 250 255Tyr Ser Arg Val Ala Arg Val Cys Lys Asn Asp Val Gly
Gly Arg Phe 260 265 270Leu Leu Glu Asp Thr Trp Thr Thr Phe Met Lys
Ala Arg Leu Asn Cys 275 280 285Ser Arg Pro Gly Glu Val Pro Phe Tyr
Tyr Asn Glu Leu Gln Ser Ala 290 295 300Phe His Leu Pro Glu Gln Asp
Leu Ile Tyr Gly Val Phe Thr Thr Asn305 310 315 320Val Asn Ser Ile
Ala Ala Ser Ala Val Cys Ala Phe Asn Leu Ser Ala 325 330 335Ile Ser
Gln Ala Phe Asn Gly Pro Phe Arg Tyr Gln Glu Asn Pro Arg 340 345
350Ala Ala Trp Leu Pro Ile Ala Asn Pro Ile Pro Asn Phe Gln Cys Gly
355 360 365Thr Leu Pro Glu Thr Gly Pro Asn Glu Asn Leu Thr Glu Arg
Ser Leu 370 375 380Gln Asp Ala Gln Arg Leu Phe Leu Met Ser Glu Ala
Val Gln Pro Val385 390 395 400Thr Pro Glu Pro Cys Val Thr Gln Asp
Ser Val Arg Phe Ser His Leu 405 410 415Val Val Asp Leu Val Gln Ala
Lys Asp Thr Leu Tyr His Val Leu Tyr 420 425 430Ile Gly Thr Glu Ser
Gly Thr Ile Leu Lys Ala Leu Ser Thr Ala Ser 435 440 445Arg Ser Leu
His Gly Cys Tyr Leu Glu Glu Leu His Val Leu Pro Pro 450 455 460Gly
Arg Arg Glu Pro Leu Arg Ser Leu Arg Ile Leu His Ser Ala Arg465 470
475 480Ala Leu Phe Val Gly Leu Arg Asp Gly Val Leu Arg Val Pro Leu
Glu 485 490 495Arg Cys Ala Ala Tyr Arg Ser Gln Gly Ala Cys Leu Gly
Ala Arg Asp 500 505 510Pro Tyr Cys Gly Trp Asp Gly Lys Gln Gln Arg
Cys Ser Thr Leu Glu 515 520 525Asp Ser Ser Asn Met Ser Leu Trp Thr
Gln Asn Ile Thr Ala Cys Pro 530 535 540Val Arg Asn Val Thr Arg Asp
Gly Gly Phe Gly Pro Trp Ser Pro Trp545 550 555 560Gln Pro Cys Glu
His Leu Asp Gly Asp Asn Ser Gly Ser Cys Leu Cys 565 570 575Arg Ala
Arg Ser Cys Asp Ser Pro Arg Pro Arg Cys Gly Gly Leu Asp 580 585
590Cys Leu Gly Pro Ala Ile His Ile Ala Asn Cys Ser Arg Asn Gly Ala
595 600 605Trp Thr Pro Trp Ser Ser Trp Ala Leu Cys Ser Thr Ser Cys
Gly Ile 610 615 620Gly Phe Gln Val Arg Gln Arg Ser Cys Ser Asn Pro
Ala Pro Arg His625 630 635 640Gly Gly Arg Ile Cys Val Gly Lys Ser
Arg Glu Glu Arg Phe Cys Asn 645 650 655Glu Asn Thr Pro Cys Pro Val
Pro Ile Phe Trp Ala Ser Trp Gly Ser 660 665 670Trp Ser Lys Cys Ser
Ser Asn Cys Gly Gly Gly Met Gln Ser Arg Arg 675 680 685Arg Ala Cys
Glu Asn Gly Asn Ser Cys Leu Gly Cys Gly Val Glu Phe 690 695 700Lys
Thr Cys Asn Pro Glu Gly Cys Pro Glu Val Arg Arg Asn Thr Pro705 710
715 720Trp Thr Pro Trp Leu Pro Val Asn Val Thr Gln Gly Gly Ala Arg
Gln 725 730 735Glu Gln Arg Phe Arg Phe Thr Cys Arg Ala Pro Leu Ala
Asp Pro His 740 745 750Gly Leu Gln Phe Gly Arg Arg Arg Thr Glu Thr
Arg Thr Cys Pro Ala 755 760 765Asp Gly Ser Gly Ser Cys Asp Thr Asp
Ala Leu Val Glu Asp Leu Leu 770 775 780Arg Ser Gly Ser Thr Ser Pro
His Thr Val Ser Gly Gly Trp Ala Ala785 790 795 800Trp Gly Pro Trp
Ser Ser Cys Ser Arg Asp Cys Glu Leu Gly Phe Arg 805 810 815Val Arg
Lys Arg Thr Cys Thr Asn Pro Glu Pro Arg Asn Gly Gly Leu 820 825
830Pro Cys Val Gly Asp Ala Ala Glu Tyr Gln Asp Cys Asn Pro Gln Ala
835 840 845Cys Pro Val Arg Gly Ala Trp Ser Cys Trp Thr Ser Trp Ser
Pro Cys 850 855 860Ser Ala Ser Cys Gly Gly Gly His Tyr Gln Arg Thr
Arg Ser Cys Thr865 870 875 880Ser Pro Ala Pro Ser Pro Gly Glu Asp
Ile Cys Leu Gly Leu His Thr 885 890 895Glu Glu Ala Leu Cys Ala Thr
Gln Ala Cys Pro Glu Gly Trp Ser Pro 900 905 910Trp Ser Glu Trp Ser
Lys Cys Thr Asp Asp Gly Ala Gln Ser Arg Ser 915 920 925Arg His Cys
Glu Glu Leu Leu Pro Gly Ser Ser Ala Cys Ala Gly Asn 930 935 940Ser
Ser Gln Ser Arg Pro Cys Pro Tyr Ser Glu Ile Pro Val Ile Leu945 950
955 960Pro Ala Ser Ser Met Glu Glu Ala Thr Gly Cys Ala Gly Phe Asn
Leu 965 970 975Ile His Leu Val Ala Thr Gly Ile Ser Cys Phe Leu Gly
Ser Gly Leu 980 985 990Leu Thr Leu Ala Val Tyr Leu Ser Cys Gln His
Cys Gln Arg Gln Ser 995 1000 1005Gln Glu Ser Thr Leu Val His Pro
Ala Thr Pro Asn His Leu His Tyr 1010 1015 1020Lys Gly Gly Gly Thr
Pro Lys Asn Glu Lys Tyr Thr Pro Met Glu Phe1025 1030 1035 1040Lys
Thr Leu Asn Lys Asn Asn Leu Ile Pro Asp Asp Arg Ala Asn Phe 1045
1050 1055Tyr Pro Leu Gln Gln Thr Asn Val Tyr Thr Thr Thr Tyr Tyr
Pro Ser 1060 1065 1070Pro Leu Asn Lys His Ser Phe Arg Pro Glu Ala
Ser Pro Gly Gln Arg 1075 1080 1085Cys Phe Pro Asn Ser
10908141PRTHomo sapiensPSCA hlg 8Met Trp Val Leu Gly Ile Ala Ala
Thr Phe Cys Gly Leu Phe Leu Leu1 5 10 15Pro Gly Phe Ala Leu Gln Ile
Gln Cys Tyr Gln Cys Glu Glu Phe Gln 20 25 30Leu Asn Asn Asp Cys Ser
Ser Pro Glu Phe Ile Val Asn Cys Thr Val 35 40 45Asn Val Gln Asp Met
Cys Gln Lys Glu Val Met Glu Gln Ser Ala Gly 50 55 60Ile Met Tyr Arg
Lys Ser Cys Ala Ser Ser Ala Ala Cys Leu Ile Ala65 70 75 80Ser Ala
Gly Tyr Gln Ser Phe Cys Ser Pro Gly Lys Leu Asn Ser Val 85 90 95Cys
Ile Ser Cys Cys Asn Thr Pro Leu Cys Asn Gly Pro Arg Pro Lys 100 105
110Lys Arg Gly Ser Ser Ala Ser Ala Leu Arg Pro Gly Leu Arg Thr Thr
115 120 125Ile Leu Phe Leu Lys Leu Ala Leu Phe Ser Ala His Cys 130
135 1409442PRTHomo sapiensETBR 9Met Gln Pro Pro Pro Ser Leu Cys Gly
Arg Ala Leu Val Ala Leu Val1 5 10 15Leu Ala Cys Gly Leu Ser Arg Ile
Trp Gly Glu Glu Arg Gly Phe Pro 20 25 30Pro Asp Arg Ala Thr Pro Leu
Leu Gln Thr Ala Glu Ile Met Thr Pro 35 40 45Pro Thr Lys Thr Leu Trp
Pro Lys Gly Ser Asn Ala Ser Leu Ala Arg 50 55 60Ser Leu Ala Pro Ala
Glu Val Pro Lys Gly Asp Arg Thr Ala Gly Ser65 70 75 80Pro Pro Arg
Thr Ile Ser Pro Pro Pro Cys Gln Gly Pro Ile Glu Ile 85 90 95Lys Glu
Thr Phe Lys Tyr Ile Asn Thr Val Val Ser Cys Leu Val Phe 100 105
110Val Leu Gly Ile Ile Gly Asn Ser Thr Leu Leu Arg Ile Ile Tyr Lys
115 120 125Asn Lys Cys Met Arg Asn Gly Pro Asn Ile Leu Ile Ala Ser
Leu Ala 130 135 140Leu Gly Asp Leu Leu His Ile Val Ile Asp Ile Pro
Ile Asn Val Tyr145 150 155 160Lys Leu Leu Ala Glu Asp Trp Pro Phe
Gly Ala Glu Met Cys Lys Leu 165 170 175Val Pro Phe Ile Gln Lys Ala
Ser Val Gly Ile Thr Val Leu Ser Leu 180 185 190Cys Ala Leu Ser Ile
Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg 195 200 205Ile Lys Gly
Ile Gly Val Pro Lys Trp Thr Ala Val Glu Ile Val Leu 210 215 220Ile
Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala Ile Gly Phe225 230
235 240Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser Tyr Leu Arg Ile Cys
Leu 245 250 255Leu His Pro Val Gln Lys Thr Ala Phe Met Gln Phe Tyr
Lys Thr Ala 260 265 270Lys Asp Trp Trp Leu Phe Ser Phe Tyr Phe Cys
Leu Pro Leu Ala Ile 275 280 285Thr Ala Phe Phe Tyr Thr Leu Met Thr
Cys Glu Met Leu Arg Lys Lys 290 295 300Ser Gly Met Gln Ile Ala Leu
Asn Asp His Leu Lys Gln Arg Arg Glu305 310 315 320Val Ala Lys Thr
Val Phe Cys Leu Val Leu Val Phe Ala Leu Cys Trp 325 330 335Leu Pro
Leu His Leu Ser Arg Ile Leu Lys Leu Thr Leu Tyr Asn Gln 340 345
350Asn Asp Pro Asn Arg Cys Glu Leu Leu Ser Phe Leu Leu Val Leu Asp
355 360 365Tyr Ile Gly Ile Asn Met Ala Ser Leu Asn Ser Cys Ile Asn
Pro Ile 370 375 380Ala Leu Tyr Leu Val Ser Lys Arg Phe Lys Asn Cys
Phe Lys Ser Cys385 390 395 400Leu Cys Cys Trp Cys Gln Ser Phe Glu
Glu Lys Gln Ser Leu Glu Glu 405 410 415Lys Gln Ser Cys Leu Lys Phe
Lys Ala Asn Asp His Gly Tyr Asp Asn 420 425 430Phe Arg Ser Ser Asn
Lys Tyr Ser Ser Ser 435 44010783PRTHomo sapiensRNF124 10Met Ser Gly
Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu1 5 10 15Leu Met
Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu Val Leu 20 25 30Ala
Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile 35 40
45Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn Leu Thr
50 55 60Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala Glu
Gly65 70 75 80Lys Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn Ala
Ser Asp Asp 85 90 95Asp Asn Leu Glu Pro Gly Phe Ile Ser Ile Val Lys
Leu Glu Ser Pro 100 105 110Arg Arg Ala Pro Arg Pro Cys Leu Ser Leu
Ala Ser Lys Ala Arg Met 115 120 125Ala Gly Glu Arg Gly Ala Ser Ala
Val Leu Phe Asp Ile Thr Glu Asp 130 135 140Arg Ala Ala Ala Glu Gln
Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro145 150 155 160Val Val Leu
Ile Trp Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val 165 170 175Tyr
Lys Asn Gln Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro 180 185
190Ala Trp Pro Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr
195 200 205Ile Phe Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys
Arg Pro 210 215 220Arg His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr
Ala Trp Ala Ile225 230 235 240Ser Gln Leu Ala Thr Arg Arg Tyr Gln
Ala Ser Cys Arg Gln Ala Arg 245 250 255Gly Glu Trp Pro Asp Ser Gly
Ser Ser Cys Ser Ser Ala Pro Val Cys 260 265 270Ala Ile Cys Leu Glu
Glu Phe Ser Glu Gly Gln Glu Leu Arg Val Ile 275 280 285Ser Cys Leu
His Glu Phe His Arg Asn Cys Val Asp Pro Trp Leu His 290 295 300Gln
His Arg Thr Cys Pro Leu Cys Val Phe Asn Ile Thr Glu Gly Asp305 310
315 320Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg Ser Tyr Gln Glu Pro
Gly 325 330 335Arg Arg Leu His Leu Ile Arg Gln His Pro Gly His Ala
His Tyr His 340 345 350Leu Pro Ala Ala Tyr Leu Leu Gly Pro Ser Arg
Ser Ala Val Ala Arg 355 360 365Pro Pro Arg Pro Gly Pro Phe Leu Pro
Ser Gln Glu Pro Gly Met Gly 370 375 380Pro Arg His His Arg Phe Pro
Arg Ala Ala His Pro Arg Ala Pro Gly385 390 395 400Glu Gln Gln Arg
Leu Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp 405 410 415Gly Met
Ser His Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro 420 425
430Val Pro Leu Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu
435 440 445Ser Tyr Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro
Ala Ser 450 455 460Asp Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser
Asp Ser Val Val465 470 475 480Asn Cys Thr Asp Ile Ser Leu Gln Gly
Val His Gly Ser Ser Ser Thr 485 490 495Phe Cys Ser Ser Leu Ser Ser
Asp Phe Asp Pro Leu Val Tyr Cys Ser 500 505 510Pro Lys Gly Asp Pro
Gln Arg Val Asp Met Gln Pro Ser Val Thr Ser 515 520 525Arg Pro Arg
Ser Leu Asp Ser Val Val Pro Thr Gly Glu Thr Gln Val 530 535 540Ser
Ser His Val His Tyr His Arg His Arg His His His Tyr Lys Lys545 550
555 560Arg Phe Gln Trp His Gly Arg Lys Pro Gly Pro Glu Thr Gly Val
Pro 565 570 575Gln Ser Arg Pro Pro Ile Pro Arg Thr Gln Pro Gln Pro
Glu Pro Pro 580 585 590Ser Pro Asp Gln Gln Val Thr Gly Ser Asn Ser
Ala Ala Pro Ser Gly 595 600 605Arg Leu Ser Asn Pro Gln Cys Pro Arg
Ala Leu Pro Glu Pro Ala Pro 610 615 620Gly Pro Val Asp Ala Ser Ser
Ile Cys Pro Ser Thr Ser Ser Leu Phe625 630 635 640Asn Leu Gln Lys
Ser Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg 645 650 655Arg Gly
Gly Pro Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala 660 665
670Thr Val His Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val
675 680 685Ala Tyr Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly
Pro Pro 690 695 700Gly Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly
Pro Cys Tyr Ser705 710 715 720Asn Ser Gln Pro Val Trp Leu Cys Leu
Thr Pro Arg Gln Pro Leu Glu 725 730 735Pro His Pro Pro Gly Glu Gly
Pro Ser Glu Trp Ser Ser Asp Thr Ala 740 745 750Glu Gly Arg Pro Cys
Pro Tyr Pro His Cys Gln Val Leu Ser Ala Gln 755 760 765Pro Gly Ser
Glu Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala Val 770 775
78011490PRTHomo sapiensSTEAP2 11Met Glu Ser Ile Ser Met Met Gly Ser
Pro Lys Ser Leu Ser Glu Thr1 5 10 15Val Leu Pro Asn Gly Ile Asn Gly
Ile Lys Asp Ala Arg Lys Val Thr 20 25 30Val Gly Val Ile Gly Ser Gly
Asp Phe Ala Lys Ser Leu Thr Ile Arg 35 40 45Leu Ile Arg Cys Gly Tyr
His Val Val Ile Gly Ser Arg Asn Pro Lys 50 55 60Phe Ala Ser Glu Phe
Phe Pro His Val Val Asp Val Thr His His Glu65 70 75 80Asp Ala Leu
Thr Lys Thr Asn Ile Ile Phe Val Ala Ile His Arg Glu 85 90 95His Tyr
Thr Ser Leu Trp Asp Leu Arg His Leu Leu Val Gly Lys Ile 100 105
110Leu Ile Asp Val Ser Asn Asn Met Arg Ile Asn Gln Tyr Pro Glu Ser
115 120 125Asn Ala Glu Tyr Leu Ala Ser Leu Phe Pro Asp Ser Leu Ile
Val Lys 130 135 140Gly Phe Asn Val Val Ser Ala Trp Ala Leu Gln Leu
Gly Pro Lys Asp145 150 155 160Ala Ser Arg Gln Val Tyr Ile Cys Ser
Asn Asn Ile Gln Ala Arg Gln 165 170 175Gln Val Ile Glu Leu Ala Arg
Gln Leu Asn Phe Ile
Pro Ile Asp Leu 180 185 190Gly Ser Leu Ser Ser Ala Arg Glu Ile Glu
Asn Leu Pro Leu Arg Leu 195 200 205Phe Thr Leu Trp Arg Gly Pro Val
Val Val Ala Ile Ser Leu Ala Thr 210 215 220Phe Phe Phe Leu Tyr Ser
Phe Val Arg Asp Val Ile His Pro Tyr Ala225 230 235 240Arg Asn Gln
Gln Ser Asp Phe Tyr Lys Ile Pro Ile Glu Ile Val Asn 245 250 255Lys
Thr Leu Pro Ile Val Ala Ile Thr Leu Leu Ser Leu Val Tyr Leu 260 265
270Ala Gly Leu Leu Ala Ala Ala Tyr Gln Leu Tyr Tyr Gly Thr Lys Tyr
275 280 285Arg Arg Phe Pro Pro Trp Leu Glu Thr Trp Leu Gln Cys Arg
Lys Gln 290 295 300Leu Gly Leu Leu Ser Phe Phe Phe Ala Met Val His
Val Ala Tyr Ser305 310 315 320Leu Cys Leu Pro Met Arg Arg Ser Glu
Arg Tyr Leu Phe Leu Asn Met 325 330 335Ala Tyr Gln Gln Val His Ala
Asn Ile Glu Asn Ser Trp Asn Glu Glu 340 345 350Glu Val Trp Arg Ile
Glu Met Tyr Ile Ser Phe Gly Ile Met Ser Leu 355 360 365Gly Leu Leu
Ser Leu Leu Ala Val Thr Ser Ile Pro Ser Val Ser Asn 370 375 380Ala
Leu Asn Trp Arg Glu Phe Ser Phe Ile Gln Ser Thr Leu Gly Tyr385 390
395 400Val Ala Leu Leu Ile Ser Thr Phe His Val Leu Ile Tyr Gly Trp
Lys 405 410 415Arg Ala Phe Glu Glu Glu Tyr Tyr Arg Phe Tyr Thr Pro
Pro Asn Phe 420 425 430Val Leu Ala Leu Val Leu Pro Ser Ile Val Ile
Leu Gly Lys Ile Ile 435 440 445Leu Phe Leu Pro Cys Ile Ser Gln Lys
Leu Lys Arg Ile Lys Lys Gly 450 455 460Trp Glu Lys Ser Gln Phe Leu
Glu Glu Gly Ile Gly Gly Thr Ile Pro465 470 475 480His Val Ser Pro
Glu Arg Val Thr Val Met 485 490121214PRTHomo sapiensTrpM4 12Met Val
Val Pro Glu Lys Glu Gln Ser Trp Ile Pro Lys Ile Phe Lys1 5 10 15Lys
Lys Thr Cys Thr Thr Phe Ile Val Asp Ser Thr Asp Pro Gly Gly 20 25
30Thr Leu Cys Gln Cys Gly Arg Pro Arg Thr Ala His Pro Ala Val Ala
35 40 45Met Glu Asp Ala Phe Gly Ala Ala Val Val Thr Val Trp Asp Ser
Asp 50 55 60Ala His Thr Thr Glu Lys Pro Thr Asp Ala Tyr Gly Glu Leu
Asp Phe65 70 75 80Thr Gly Ala Gly Arg Lys His Ser Asn Phe Leu Arg
Leu Ser Asp Arg 85 90 95Thr Asp Pro Ala Ala Val Tyr Ser Leu Val Thr
Arg Thr Trp Gly Phe 100 105 110Arg Ala Pro Asn Leu Val Val Ser Val
Leu Gly Gly Ser Gly Gly Pro 115 120 125Val Leu Gln Thr Trp Leu Gln
Asp Leu Leu Arg Arg Gly Leu Val Arg 130 135 140Ala Ala Gln Ser Thr
Gly Ala Trp Ile Val Thr Gly Gly Leu His Thr145 150 155 160Gly Ile
Gly Arg His Val Gly Val Ala Val Arg Asp His Gln Met Ala 165 170
175Ser Thr Gly Gly Thr Lys Val Val Ala Met Gly Val Ala Pro Trp Gly
180 185 190Val Val Arg Asn Arg Asp Thr Leu Ile Asn Pro Lys Gly Ser
Phe Pro 195 200 205Ala Arg Tyr Arg Trp Arg Gly Asp Pro Glu Asp Gly
Val Gln Phe Pro 210 215 220Leu Asp Tyr Asn Tyr Ser Ala Phe Phe Leu
Val Asp Asp Gly Thr His225 230 235 240Gly Cys Leu Gly Gly Glu Asn
Arg Phe Arg Leu Arg Leu Glu Ser Tyr 245 250 255Ile Ser Gln Gln Lys
Thr Gly Val Gly Gly Thr Gly Ile Asp Ile Pro 260 265 270Val Leu Leu
Leu Leu Ile Asp Gly Asp Glu Lys Met Leu Thr Arg Ile 275 280 285Glu
Asn Ala Thr Gln Ala Gln Leu Pro Cys Leu Leu Val Ala Gly Ser 290 295
300Gly Gly Ala Ala Asp Cys Leu Ala Glu Thr Leu Glu Asp Thr Leu
Ala305 310 315 320Pro Gly Ser Gly Gly Ala Arg Gln Gly Glu Ala Arg
Asp Arg Ile Arg 325 330 335Arg Phe Phe Pro Lys Gly Asp Leu Glu Val
Leu Gln Ala Gln Val Glu 340 345 350Arg Ile Met Thr Arg Lys Glu Leu
Leu Thr Val Tyr Ser Ser Glu Asp 355 360 365Gly Ser Glu Glu Phe Glu
Thr Ile Val Leu Lys Ala Leu Val Lys Ala 370 375 380Cys Gly Ser Ser
Glu Ala Ser Ala Tyr Leu Asp Glu Leu Arg Leu Ala385 390 395 400Val
Ala Trp Asn Arg Val Asp Ile Ala Gln Ser Glu Leu Phe Arg Gly 405 410
415Asp Ile Gln Trp Arg Ser Phe His Leu Glu Ala Ser Leu Met Asp Ala
420 425 430Leu Leu Asn Asp Arg Pro Glu Phe Val Arg Leu Leu Ile Ser
His Gly 435 440 445Leu Ser Leu Gly His Phe Leu Thr Pro Met Arg Leu
Ala Gln Leu Tyr 450 455 460Ser Ala Ala Pro Ser Asn Ser Leu Ile Arg
Asn Leu Leu Asp Gln Ala465 470 475 480Ser His Ser Ala Gly Thr Lys
Ala Pro Ala Leu Lys Gly Gly Ala Ala 485 490 495Glu Leu Arg Pro Pro
Asp Val Gly His Val Leu Arg Met Leu Leu Gly 500 505 510Lys Met Cys
Ala Pro Arg Tyr Pro Ser Gly Gly Ala Trp Asp Pro His 515 520 525Pro
Gly Gln Gly Phe Gly Glu Ser Met Tyr Leu Leu Ser Asp Lys Ala 530 535
540Thr Ser Pro Leu Ser Leu Asp Ala Gly Leu Gly Gln Ala Pro Trp
Ser545 550 555 560Asp Leu Leu Leu Trp Ala Leu Leu Leu Asn Arg Ala
Gln Met Ala Met 565 570 575Tyr Phe Trp Glu Met Gly Ser Asn Ala Val
Ser Ser Ala Leu Gly Ala 580 585 590Cys Leu Leu Leu Arg Val Met Ala
Arg Leu Glu Pro Asp Ala Glu Glu 595 600 605Ala Ala Arg Arg Lys Asp
Leu Ala Phe Lys Phe Glu Gly Met Gly Val 610 615 620Asp Leu Phe Gly
Glu Cys Tyr Arg Ser Ser Glu Val Arg Ala Ala Arg625 630 635 640Leu
Leu Leu Arg Arg Cys Pro Leu Trp Gly Asp Ala Thr Cys Leu Gln 645 650
655Leu Ala Met Gln Ala Asp Ala Arg Ala Phe Phe Ala Gln Asp Gly Val
660 665 670Gln Ser Leu Leu Thr Gln Lys Trp Trp Gly Asp Met Ala Ser
Thr Thr 675 680 685Pro Ile Trp Ala Leu Val Leu Ala Phe Phe Cys Pro
Pro Leu Ile Tyr 690 695 700Thr Arg Leu Ile Thr Phe Arg Lys Ser Glu
Glu Glu Pro Thr Arg Glu705 710 715 720Glu Leu Glu Phe Asp Met Asp
Ser Val Ile Asn Gly Glu Gly Pro Val 725 730 735Gly Thr Ala Asp Pro
Ala Glu Lys Thr Pro Leu Gly Val Pro Arg Gln 740 745 750Ser Gly Arg
Pro Gly Cys Cys Gly Gly Arg Cys Gly Gly Arg Arg Cys 755 760 765Leu
Arg Arg Trp Phe His Phe Trp Gly Ala Pro Val Thr Ile Phe Met 770 775
780Gly Asn Val Val Ser Tyr Leu Leu Phe Leu Leu Leu Phe Ser Arg
Val785 790 795 800Leu Leu Val Asp Phe Gln Pro Ala Pro Pro Gly Ser
Leu Glu Leu Leu 805 810 815Leu Tyr Phe Trp Ala Phe Thr Leu Leu Cys
Glu Glu Leu Arg Gln Gly 820 825 830Leu Ser Gly Gly Gly Gly Ser Leu
Ala Ser Gly Gly Pro Gly Pro Gly 835 840 845His Ala Ser Leu Ser Gln
Arg Leu Arg Leu Tyr Leu Ala Asp Ser Trp 850 855 860Asn Gln Cys Asp
Leu Val Ala Leu Thr Cys Phe Leu Leu Gly Val Gly865 870 875 880Cys
Arg Leu Thr Pro Gly Leu Tyr His Leu Gly Arg Thr Val Leu Cys 885 890
895Ile Asp Phe Met Val Phe Thr Val Arg Leu Leu His Ile Phe Thr Val
900 905 910Asn Lys Gln Leu Gly Pro Lys Ile Val Ile Val Ser Lys Met
Met Lys 915 920 925Asp Val Phe Phe Phe Leu Phe Phe Leu Gly Val Trp
Leu Val Ala Tyr 930 935 940Gly Val Ala Thr Glu Gly Leu Leu Arg Pro
Arg Asp Ser Asp Phe Pro945 950 955 960Ser Ile Leu Arg Arg Val Phe
Tyr Arg Pro Tyr Leu Gln Ile Phe Gly 965 970 975Gln Ile Pro Gln Glu
Asp Met Asp Val Ala Leu Met Glu His Ser Asn 980 985 990Cys Ser Ser
Glu Pro Gly Phe Trp Ala His Pro Pro Gly Ala Gln Ala 995 1000
1005Gly Thr Cys Val Ser Gln Tyr Ala Asn Trp Leu Val Val Leu Leu Leu
1010 1015 1020Val Ile Phe Leu Leu Val Ala Asn Ile Leu Leu Val Asn
Leu Leu Ile1025 1030 1035 1040Ala Met Phe Ser Tyr Thr Phe Gly Lys
Val Gln Gly Asn Ser Asp Leu 1045 1050 1055Tyr Trp Lys Ala Gln Arg
Tyr Arg Leu Ile Arg Glu Phe His Ser Arg 1060 1065 1070Pro Ala Leu
Ala Pro Pro Phe Ile Val Ile Ser His Leu Arg Leu Leu 1075 1080
1085Leu Arg Gln Leu Cys Arg Arg Pro Arg Ser Pro Gln Pro Ser Ser Pro
1090 1095 1100Ala Leu Glu His Phe Arg Val Tyr Leu Ser Lys Glu Ala
Glu Arg Lys1105 1110 1115 1120Leu Leu Thr Trp Glu Ser Val His Lys
Glu Asn Phe Leu Leu Ala Arg 1125 1130 1135Ala Arg Asp Lys Arg Glu
Ser Asp Ser Glu Arg Leu Lys Arg Thr Ser 1140 1145 1150Gln Lys Val
Asp Leu Ala Leu Lys Gln Leu Gly His Ile Arg Glu Tyr 1155 1160
1165Glu Gln Arg Leu Lys Val Leu Glu Arg Glu Val Gln Gln Cys Ser Arg
1170 1175 1180Val Leu Gly Trp Val Ala Glu Ala Leu Ser Arg Ser Ala
Leu Leu Pro1185 1190 1195 1200Pro Gly Gly Pro Pro Pro Pro Asp Leu
Pro Gly Ser Lys Asp 1205 121013188PRTHomo sapiensTDGF1 13Met Asp
Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp Ile1 5 10 15Met
Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly Leu Gly 20 25
30His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala Phe Arg Asp
35 40 45Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg Pro Arg Ser
Ser 50 55 60Gln Arg Val Pro Pro Met Gly Ile Gln His Ser Lys Glu Leu
Asn Arg65 70 75 80Thr Cys Cys Leu Asn Gly Gly Thr Cys Met Leu Gly
Ser Phe Cys Ala 85 90 95Cys Pro Pro Ser Phe Tyr Gly Arg Asn Cys Glu
His Asp Val Arg Lys 100 105 110Glu Asn Cys Gly Ser Val Pro His Asp
Thr Trp Leu Pro Lys Lys Cys 115 120 125Ser Leu Cys Lys Cys Trp His
Gly Gln Leu Arg Cys Phe Pro Gln Ala 130 135 140Phe Leu Pro Gly Cys
Asp Gly Leu Val Met Asp Glu His Leu Val Ala145 150 155 160Ser Arg
Thr Pro Glu Leu Pro Pro Ser Ala Arg Thr Thr Thr Phe Met 165 170
175Leu Val Gly Ile Cys Leu Ser Ile Gln Ser Tyr Tyr 180
185141033PRTHomo sapiensCD21 14Met Gly Ala Ala Gly Leu Leu Gly Val
Phe Leu Ala Leu Val Ala Pro1 5 10 15Gly Val Leu Gly Ile Ser Cys Gly
Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30Arg Ile Ser Tyr Tyr Ser Thr
Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45Tyr Ser Cys Ser Gly Thr
Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60Cys Ile Thr Lys Asp
Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro65 70 75 80Lys Cys Glu
Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95Pro Gly
Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105
110Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys
115 120 125Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg
Leu Pro 130 135 140Thr Cys Val Ser Val Phe Pro Leu Glu Cys Pro Ala
Leu Pro Met Ile145 150 155 160His Asn Gly His His Thr Ser Glu Asn
Val Gly Ser Ile Ala Pro Gly 165 170 175Leu Ser Val Thr Tyr Ser Cys
Glu Ser Gly Tyr Leu Leu Val Gly Glu 180 185 190Lys Ile Ile Asn Cys
Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro 195 200 205Thr Cys Glu
Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly 210 215 220Lys
Val Lys Glu Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe225 230
235 240Phe Cys Asp Glu Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg
Cys 245 250 255Val Ile Ala Gly Gln Gly Val Ala Trp Thr Lys Met Pro
Val Cys Glu 260 265 270Glu Ile Phe Cys Pro Ser Pro Pro Pro Ile Leu
Asn Gly Arg His Ile 275 280 285Gly Asn Ser Leu Ala Asn Val Ser Tyr
Gly Ser Ile Val Thr Tyr Thr 290 295 300Cys Asp Pro Asp Pro Glu Glu
Gly Val Asn Phe Ile Leu Ile Gly Glu305 310 315 320Ser Thr Leu Arg
Cys Thr Val Asp Ser Gln Lys Thr Gly Thr Trp Ser 325 330 335Gly Pro
Ala Pro Arg Cys Glu Leu Ser Thr Ser Ala Val Gln Cys Pro 340 345
350His Pro Gln Ile Leu Arg Gly Arg Met Val Ser Gly Gln Lys Asp Arg
355 360 365Tyr Thr Tyr Asn Asp Thr Val Ile Phe Ala Cys Met Phe Gly
Phe Thr 370 375 380Leu Lys Gly Ser Lys Gln Ile Arg Cys Asn Ala Gln
Gly Thr Trp Glu385 390 395 400Pro Ser Ala Pro Val Cys Glu Lys Glu
Cys Gln Ala Pro Pro Asn Ile 405 410 415Leu Asn Gly Gln Lys Glu Asp
Arg His Met Val Arg Phe Asp Pro Gly 420 425 430Thr Ser Ile Lys Tyr
Ser Cys Asn Pro Gly Tyr Val Leu Val Gly Glu 435 440 445Glu Ser Ile
Gln Cys Thr Ser Glu Gly Val Trp Thr Pro Pro Val Pro 450 455 460Gln
Cys Lys Val Ala Ala Cys Glu Ala Thr Gly Arg Gln Leu Leu Thr465 470
475 480Lys Pro Gln His Gln Phe Val Arg Pro Asp Val Asn Ser Ser Cys
Gly 485 490 495Glu Gly Tyr Lys Leu Ser Gly Ser Val Tyr Gln Glu Cys
Gln Gly Thr 500 505 510Ile Pro Trp Phe Met Glu Ile Arg Leu Cys Lys
Glu Ile Thr Cys Pro 515 520 525Pro Pro Pro Val Ile Tyr Asn Gly Ala
His Thr Gly Ser Ser Leu Glu 530 535 540Asp Phe Pro Tyr Gly Thr Thr
Val Thr Tyr Thr Cys Asn Pro Gly Pro545 550 555 560Glu Arg Gly Val
Glu Phe Ser Leu Ile Gly Glu Ser Thr Ile Arg Cys 565 570 575Thr Ser
Asn Asp Gln Glu Arg Gly Thr Trp Ser Gly Pro Ala Pro Leu 580 585
590Cys Lys Leu Ser Leu Leu Ala Val Gln Cys Ser His Val His Ile Ala
595 600 605Asn Gly Tyr Lys Ile Ser Gly Lys Glu Ala Pro Tyr Phe Tyr
Asn Asp 610 615 620Thr Val Thr Phe Lys Cys Tyr Ser Gly Phe Thr Leu
Lys Gly Ser Ser625 630 635 640Gln Ile Arg Cys Lys Ala Asp Asn Thr
Trp Asp Pro Glu Ile Pro Val 645 650 655Cys Glu Lys Glu Thr Cys Gln
His Val Arg Gln Ser Leu Gln Glu Leu 660 665 670Pro Ala Gly Ser Arg
Val Glu Leu Val Asn Thr Ser Cys Gln Asp Gly 675 680 685Tyr Gln Leu
Thr Gly His Ala Tyr Gln Met Cys Gln Asp Ala Glu Asn 690 695 700Gly
Ile Trp Phe Lys Lys Ile Pro Leu Cys Lys Val Ile His Cys His705 710
715 720Pro Pro Pro Val Ile Val Asn Gly Lys His Thr Gly Met Met Ala
Glu 725 730 735Asn Phe Leu Tyr Gly Asn Glu Val Ser Tyr Glu Cys Asp
Gln Gly Phe 740 745 750Tyr Leu Leu Gly Glu
Lys Lys Leu Gln Cys Arg Ser Asp Ser Lys Gly 755 760 765His Gly Ser
Trp Ser Gly Pro Ser Pro Gln Cys Leu Arg Ser Pro Pro 770 775 780Val
Thr Arg Cys Pro Asn Pro Glu Val Lys His Gly Tyr Lys Leu Asn785 790
795 800Lys Thr His Ser Ala Tyr Ser His Asn Asp Ile Val Tyr Val Asp
Cys 805 810 815Asn Pro Gly Phe Ile Met Asn Gly Ser Arg Val Ile Arg
Cys His Thr 820 825 830Asp Asn Thr Trp Val Pro Gly Val Pro Thr Cys
Ile Lys Lys Ala Phe 835 840 845Ile Gly Cys Pro Pro Pro Pro Lys Thr
Pro Asn Gly Asn His Thr Gly 850 855 860Gly Asn Ile Ala Arg Phe Ser
Pro Gly Met Ser Ile Leu Tyr Ser Cys865 870 875 880Asp Gln Gly Tyr
Leu Leu Val Gly Glu Ala Leu Leu Leu Cys Thr His 885 890 895Glu Gly
Thr Trp Ser Gln Pro Ala Pro His Cys Lys Glu Val Asn Cys 900 905
910Ser Ser Pro Ala Asp Met Asp Gly Ile Gln Lys Gly Leu Glu Pro Arg
915 920 925Lys Met Tyr Gln Tyr Gly Ala Val Val Thr Leu Glu Cys Glu
Asp Gly 930 935 940Tyr Met Leu Glu Gly Ser Pro Gln Ser Gln Cys Gln
Ser Asp His Gln945 950 955 960Trp Asn Pro Pro Leu Ala Val Cys Arg
Ser Arg Ser Leu Ala Pro Val 965 970 975Leu Cys Gly Ile Ala Ala Gly
Leu Ile Leu Leu Thr Phe Leu Ile Val 980 985 990Ile Thr Leu Tyr Val
Ile Ser Lys His Arg Glu Arg Asn Tyr Tyr Thr 995 1000 1005Asp Thr
Ser Gln Lys Glu Ala Phe His Leu Glu Ala Arg Glu Val Tyr 1010 1015
1020Ser Val Asp Pro Tyr Asn Pro Ala Ser1025 103015229PRTHomo
sapiensCD79B 15Met Ala Arg Leu Ala Leu Ser Pro Val Pro Ser His Trp
Met Val Ala1 5 10 15Leu Leu Leu Leu Leu Ser Ala Glu Pro Val Pro Ala
Ala Arg Ser Glu 20 25 30Asp Arg Tyr Arg Asn Pro Lys Gly Ser Ala Cys
Ser Arg Ile Trp Gln 35 40 45Ser Pro Arg Phe Ile Ala Arg Lys Arg Gly
Phe Thr Val Lys Met His 50 55 60Cys Tyr Met Asn Ser Ala Ser Gly Asn
Val Ser Trp Leu Trp Lys Gln65 70 75 80Glu Met Asp Glu Asn Pro Gln
Gln Leu Lys Leu Glu Lys Gly Arg Met 85 90 95Glu Glu Ser Gln Asn Glu
Ser Leu Ala Thr Leu Thr Ile Gln Gly Ile 100 105 110Arg Phe Glu Asp
Asn Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn 115 120 125Thr Ser
Glu Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly 130 135
140Phe Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp
Gly145 150 155 160Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe
Ile Ile Val Pro 165 170 175Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser
Lys Ala Gly Met Glu Glu 180 185 190Asp His Thr Tyr Glu Gly Leu Asp
Ile Asp Gln Thr Ala Thr Tyr Glu 195 200 205Asp Ile Val Thr Leu Arg
Thr Gly Glu Val Lys Trp Ser Val Gly Glu 210 215 220His Pro Gly Gln
Glu22516508PRTHomo sapiensFcRH2 16Met Leu Leu Trp Ser Leu Leu Val
Ile Phe Asp Ala Val Thr Glu Gln1 5 10 15Ala Asp Ser Leu Thr Leu Val
Ala Pro Ser Ser Val Phe Glu Gly Asp 20 25 30Ser Ile Val Leu Lys Cys
Gln Gly Glu Gln Asn Trp Lys Ile Gln Lys 35 40 45Met Ala Tyr His Lys
Asp Asn Lys Glu Leu Ser Val Phe Lys Lys Phe 50 55 60Ser Asp Phe Leu
Ile Gln Ser Ala Val Leu Ser Asp Ser Gly Asn Tyr65 70 75 80Phe Cys
Ser Thr Lys Gly Gln Leu Phe Leu Trp Asp Lys Thr Ser Asn 85 90 95Ile
Val Lys Ile Lys Val Gln Glu Leu Phe Gln Arg Pro Val Leu Thr 100 105
110Ala Ser Ser Phe Gln Pro Ile Glu Gly Gly Pro Val Ser Leu Lys Cys
115 120 125Glu Thr Arg Leu Ser Pro Gln Arg Leu Asp Val Gln Leu Gln
Phe Cys 130 135 140Phe Phe Arg Glu Asn Gln Val Leu Gly Ser Gly Trp
Ser Ser Ser Pro145 150 155 160Glu Leu Gln Ile Ser Ala Val Trp Ser
Glu Asp Thr Gly Ser Tyr Trp 165 170 175Cys Lys Ala Glu Thr Val Thr
His Arg Ile Arg Lys Gln Ser Leu Gln 180 185 190Ser Gln Ile His Val
Gln Arg Ile Pro Ile Ser Asn Val Ser Leu Glu 195 200 205Ile Arg Ala
Pro Gly Gly Gln Val Thr Glu Gly Gln Lys Leu Ile Leu 210 215 220Leu
Cys Ser Val Ala Gly Gly Thr Gly Asn Val Thr Phe Ser Trp Tyr225 230
235 240Arg Glu Ala Thr Gly Thr Ser Met Gly Lys Lys Thr Gln Arg Ser
Leu 245 250 255Ser Ala Glu Leu Glu Ile Pro Ala Val Lys Glu Ser Asp
Ala Gly Lys 260 265 270Tyr Tyr Cys Arg Ala Asp Asn Gly His Val Pro
Ile Gln Ser Lys Val 275 280 285Val Asn Ile Pro Val Arg Ile Pro Val
Ser Arg Pro Val Leu Thr Leu 290 295 300Arg Ser Pro Gly Ala Gln Ala
Ala Val Gly Asp Leu Leu Glu Leu His305 310 315 320Cys Glu Ala Leu
Arg Gly Ser Pro Pro Ile Leu Tyr Gln Phe Tyr His 325 330 335Glu Asp
Val Thr Leu Gly Asn Ser Ser Ala Pro Ser Gly Gly Gly Ala 340 345
350Ser Phe Asn Leu Ser Leu Thr Ala Glu His Ser Gly Asn Tyr Ser Cys
355 360 365Glu Ala Asn Asn Gly Leu Gly Ala Gln Cys Ser Glu Ala Val
Pro Val 370 375 380Ser Ile Ser Gly Pro Asp Gly Tyr Arg Arg Asp Leu
Met Thr Ala Gly385 390 395 400Val Leu Trp Gly Leu Phe Gly Val Leu
Gly Phe Thr Gly Val Ala Leu 405 410 415Leu Leu Tyr Ala Leu Phe His
Lys Ile Ser Gly Glu Ser Ser Ala Thr 420 425 430Asn Glu Pro Arg Gly
Ala Ser Arg Pro Asn Pro Gln Glu Phe Thr Tyr 435 440 445Ser Ser Pro
Thr Pro Asp Met Glu Glu Leu Gln Pro Val Tyr Val Asn 450 455 460Val
Gly Ser Val Asp Val Asp Val Val Tyr Ser Gln Val Trp Ser Met465 470
475 480Gln Gln Pro Glu Ser Ser Ala Asn Ile Arg Thr Leu Leu Glu Asn
Lys 485 490 495Asp Ser Gln Val Ile Tyr Ser Ser Val Lys Lys Ser 500
505171255PRTHomo sapiensHER2 17Met Glu Leu Ala Ala Leu Cys Arg Trp
Gly Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln
Val Cys Thr Gly Thr Asp Met Lys 20 25 30Leu Arg Leu Pro Ala Ser Pro
Glu Thr His Leu Asp Met Leu Arg His 35 40 45Leu Tyr Gln Gly Cys Gln
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60Leu Pro Thr Asn Ala
Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr
Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95Gln Arg
Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105
110Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro
115 120 125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu
Arg Ser 130 135 140Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln
Arg Asn Pro Gln145 150 155 160Leu Cys Tyr Gln Asp Thr Ile Leu Trp
Lys Asp Ile Phe His Lys Asn 165 170 175Asn Gln Leu Ala Leu Thr Leu
Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190His Pro Cys Ser Pro
Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205Ser Glu Asp
Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220Ala
Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys225 230
235 240Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys
Leu 245 250 255His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro
Ala Leu Val 260 265 270Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro
Asn Pro Glu Gly Arg 275 280 285Tyr Thr Phe Gly Ala Ser Cys Val Thr
Ala Cys Pro Tyr Asn Tyr Leu 290 295 300Ser Thr Asp Val Gly Ser Cys
Thr Leu Val Cys Pro Leu His Asn Gln305 310 315 320Glu Val Thr Ala
Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335Pro Cys
Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345
350Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys
355 360 365Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp
Gly Asp 370 375 380Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln
Leu Gln Val Phe385 390 395 400Glu Thr Leu Glu Glu Ile Thr Gly Tyr
Leu Tyr Ile Ser Ala Trp Pro 405 410 415Asp Ser Leu Pro Asp Leu Ser
Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430Gly Arg Ile Leu His
Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445Gly Ile Ser
Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460Leu
Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val465 470
475 480Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His
Thr 485 490 495Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu
Ala Cys His 500 505 510Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro
Gly Pro Thr Gln Cys 515 520 525Val Asn Cys Ser Gln Phe Leu Arg Gly
Gln Glu Cys Val Glu Glu Cys 530 535 540Arg Val Leu Gln Gly Leu Pro
Arg Glu Tyr Val Asn Ala Arg His Cys545 550 555 560Leu Pro Cys His
Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575Phe Gly
Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585
590Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu
595 600 605Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala
Cys Gln 610 615 620Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp
Leu Asp Asp Lys625 630 635 640Gly Cys Pro Ala Glu Gln Arg Ala Ser
Pro Leu Thr Ser Ile Ile Ser 645 650 655Ala Val Val Gly Ile Leu Leu
Val Val Val Leu Gly Val Val Phe Gly 660 665 670Ile Leu Ile Lys Arg
Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685Arg Leu Leu
Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700Ala
Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu705 710
715 720Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr
Lys 725 730 735Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro
Val Ala Ile 740 745 750Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala
Asn Lys Glu Ile Leu 755 760 765Asp Glu Ala Tyr Val Met Ala Gly Val
Gly Ser Pro Tyr Val Ser Arg 770 775 780Leu Leu Gly Ile Cys Leu Thr
Ser Thr Val Gln Leu Val Thr Gln Leu785 790 795 800Met Pro Tyr Gly
Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815Leu Gly
Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825
830Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala
835 840 845Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr
Asp Phe 850 855 860Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu
Tyr His Ala Asp865 870 875 880Gly Gly Lys Val Pro Ile Lys Trp Met
Ala Leu Glu Ser Ile Leu Arg 885 890 895Arg Arg Phe Thr His Gln Ser
Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910Trp Glu Leu Met Thr
Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925Arg Glu Ile
Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940Pro
Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met945 950
955 960Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu
Phe 965 970 975Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile
Gln Asn Glu 980 985 990Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr
Phe Tyr Arg Ser Leu 995 1000 1005Leu Glu Asp Asp Asp Met Gly Asp
Leu Val Asp Ala Glu Glu Tyr Leu 1010 1015 1020Val Pro Gln Gln Gly
Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly1025 1030 1035 1040Gly
Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly 1045
1050 1055Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala
Pro Arg 1060 1065 1070Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser
Asp Val Phe Asp Gly 1075 1080 1085Asp Leu Gly Met Gly Ala Ala Lys
Gly Leu Gln Ser Leu Pro Thr His 1090 1095 1100Asp Pro Ser Pro Leu
Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu1105 1110 1115 1120Pro
Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln 1125
1130 1135Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro
Ser Pro 1140 1145 1150Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala
Gly Ala Thr Leu Glu 1155 1160 1165Arg Pro Lys Thr Leu Ser Pro Gly
Lys Asn Gly Val Val Lys Asp Val 1170 1175 1180Phe Ala Phe Gly Gly
Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln1185 1190 1195 1200Gly
Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala 1205
1210 1215Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg
Gly Ala 1220 1225 1230Pro Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala
Glu Asn Pro Glu Tyr 1235 1240 1245Leu Gly Leu Asp Val Pro Val 1250
125518344PRTHomo sapiensCEACAM6 18Met Gly Pro Pro Ser Ala Pro Pro
Cys Arg Leu His Val Pro Trp Lys1 5 10 15Glu Val Leu Leu Thr Ala Ser
Leu Leu Thr Phe Trp Asn Pro Pro Thr 20 25 30Thr Ala Lys Leu Thr Ile
Glu Ser Thr Pro Phe Asn Val Ala Glu Gly 35 40 45Lys Glu Val Leu Leu
Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly 50 55 60Tyr Ser Trp Tyr
Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val65 70 75 80Gly Tyr
Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser 85 90 95Gly
Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val 100 105
110Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp
115 120 125Leu Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro
Glu Leu 130 135 140Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro
Val Glu Asp Lys145 150 155 160Asp Ala Val Ala Phe Thr Cys Glu Pro
Glu Val Gln Asn Thr Thr Tyr 165 170 175Leu Trp Trp Val Asn Gly Gln
Ser Leu Pro Val Ser Pro Arg Leu Gln 180 185 190Leu
Ser Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn 195 200
205Asp Ala Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn
210 215 220Arg Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp
Val Pro225 230 235 240Thr Ile Ser Pro Ser Lys Ala Asn Tyr Arg Pro
Gly Glu Asn Leu Asn 245 250 255Leu Ser Cys His Ala Ala Ser Asn Pro
Pro Ala Gln Tyr Ser Trp Phe 260 265 270Ile Asn Gly Thr Phe Gln Gln
Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280 285Ile Thr Val Asn Asn
Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser 290 295 300Ala Thr Gly
Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly305 310 315
320Ser Ala Pro Val Leu Ser Ala Val Ala Thr Val Gly Ile Thr Ile Gly
325 330 335Val Leu Ala Arg Val Ala Leu Ile 34019411PRTHomo
sapiensDPEP1 19Met Trp Ser Gly Trp Trp Leu Trp Pro Leu Val Ala Val
Cys Thr Ala1 5 10 15Asp Phe Phe Arg Asp Glu Ala Glu Arg Ile Met Arg
Asp Ser Pro Val 20 25 30Ile Asp Gly His Asn Asp Leu Pro Trp Gln Leu
Leu Asp Met Phe Asn 35 40 45Asn Arg Leu Gln Asp Glu Arg Ala Asn Leu
Thr Thr Leu Ala Gly Thr 50 55 60His Thr Asn Ile Pro Lys Leu Arg Ala
Gly Phe Val Gly Gly Gln Phe65 70 75 80Trp Ser Val Tyr Thr Pro Cys
Asp Thr Gln Asn Lys Asp Ala Val Arg 85 90 95Arg Thr Leu Glu Gln Met
Asp Val Val His Arg Met Cys Arg Met Tyr 100 105 110Pro Glu Thr Phe
Leu Tyr Val Thr Ser Ser Ala Gly Ile Arg Gln Ala 115 120 125Phe Arg
Glu Gly Lys Val Ala Ser Leu Ile Gly Val Glu Gly Gly His 130 135
140Ser Ile Asp Ser Ser Leu Gly Val Leu Arg Ala Leu Tyr Gln Leu
Gly145 150 155 160Met Arg Tyr Leu Thr Leu Thr His Ser Cys Asn Thr
Pro Trp Ala Asp 165 170 175Asn Trp Leu Val Asp Thr Gly Asp Ser Glu
Pro Gln Ser Gln Gly Leu 180 185 190Ser Pro Phe Gly Gln Arg Val Val
Lys Glu Leu Asn Arg Leu Gly Val 195 200 205Leu Ile Asp Leu Ala His
Val Ser Val Ala Thr Met Lys Ala Thr Leu 210 215 220Gln Leu Ser Arg
Ala Pro Val Ile Phe Ser His Ser Ser Ala Tyr Ser225 230 235 240Val
Cys Ala Ser Arg Arg Asn Val Pro Asp Asp Val Leu Arg Leu Val 245 250
255Lys Gln Thr Asp Ser Leu Val Met Val Asn Phe Tyr Asn Asn Tyr Ile
260 265 270Ser Cys Thr Asn Lys Ala Asn Leu Ser Gln Val Ala Asp His
Leu Asp 275 280 285His Ile Lys Glu Val Ala Gly Ala Arg Ala Val Gly
Phe Gly Gly Asp 290 295 300Phe Asp Gly Val Pro Arg Val Pro Glu Gly
Leu Glu Asp Val Ser Lys305 310 315 320Tyr Pro Asp Leu Ile Ala Glu
Leu Leu Arg Arg Asn Trp Thr Glu Ala 325 330 335Glu Val Lys Gly Ala
Leu Ala Asp Asn Leu Leu Arg Val Phe Glu Ala 340 345 350Val Glu Gln
Ala Ser Asn Leu Thr Gln Ala Pro Glu Glu Glu Pro Ile 355 360 365Pro
Leu Asp Gln Leu Gly Gly Ser Cys Arg Thr His Tyr Gly Tyr Ser 370 375
380Ser Gly Ala Ser Ser Leu His Arg His Trp Gly Leu Leu Leu Ala
Ser385 390 395 400Leu Ala Pro Leu Val Leu Cys Leu Ser Leu Leu 405
41020553PRTHomo sapiensIL20Ra 20Met Arg Ala Pro Gly Arg Pro Ala Leu
Arg Pro Leu Pro Leu Pro Pro1 5 10 15Leu Leu Leu Leu Leu Leu Ala Ala
Pro Trp Gly Arg Ala Val Pro Cys 20 25 30Val Ser Gly Gly Leu Pro Lys
Pro Ala Asn Ile Thr Phe Leu Ser Ile 35 40 45Asn Met Lys Asn Val Leu
Gln Trp Thr Pro Pro Glu Gly Leu Gln Gly 50 55 60Val Lys Val Thr Tyr
Thr Val Gln Tyr Phe Ile Tyr Gly Gln Lys Lys65 70 75 80Trp Leu Asn
Lys Ser Glu Cys Arg Asn Ile Asn Arg Thr Tyr Cys Asp 85 90 95Leu Ser
Ala Glu Thr Ser Asp Tyr Glu His Gln Tyr Tyr Ala Lys Val 100 105
110Lys Ala Ile Trp Gly Thr Lys Cys Ser Lys Trp Ala Glu Ser Gly Arg
115 120 125Phe Tyr Pro Phe Leu Glu Thr Gln Ile Gly Pro Pro Glu Val
Ala Leu 130 135 140Thr Thr Asp Glu Lys Ser Ile Ser Val Val Leu Thr
Ala Pro Glu Lys145 150 155 160Trp Lys Arg Asn Pro Glu Asp Leu Pro
Val Ser Met Gln Gln Ile Tyr 165 170 175Ser Asn Leu Lys Tyr Asn Val
Ser Val Leu Asn Thr Lys Ser Asn Arg 180 185 190Thr Trp Ser Gln Cys
Val Thr Asn His Thr Leu Val Leu Thr Trp Leu 195 200 205Glu Pro Asn
Thr Leu Tyr Cys Val His Val Glu Ser Phe Val Pro Gly 210 215 220Pro
Pro Arg Arg Ala Gln Pro Ser Glu Lys Gln Cys Ala Arg Thr Leu225 230
235 240Lys Asp Gln Ser Ser Glu Phe Lys Ala Lys Ile Ile Phe Trp Tyr
Val 245 250 255Leu Pro Ile Ser Ile Thr Val Phe Leu Phe Ser Val Met
Gly Tyr Ser 260 265 270Ile Tyr Arg Tyr Ile His Val Gly Lys Glu Lys
His Pro Ala Asn Leu 275 280 285Ile Leu Ile Tyr Gly Asn Glu Phe Asp
Lys Arg Phe Phe Val Pro Ala 290 295 300Glu Lys Ile Val Ile Asn Phe
Ile Thr Leu Asn Ile Ser Asp Asp Ser305 310 315 320Lys Ile Ser His
Gln Asp Met Ser Leu Leu Gly Lys Ser Ser Asp Val 325 330 335Ser Ser
Leu Asn Asp Pro Gln Pro Ser Gly Asn Leu Arg Pro Pro Gln 340 345
350Glu Glu Glu Glu Val Lys His Leu Gly Tyr Ala Ser His Leu Met Glu
355 360 365Ile Phe Cys Asp Ser Glu Glu Asn Thr Glu Gly Thr Ser Phe
Thr Gln 370 375 380Gln Glu Ser Leu Ser Arg Thr Ile Pro Pro Asp Lys
Thr Val Ile Glu385 390 395 400Tyr Glu Tyr Asp Val Arg Thr Thr Asp
Ile Cys Ala Gly Pro Glu Glu 405 410 415Gln Glu Leu Ser Leu Gln Glu
Glu Val Ser Thr Gln Gly Thr Leu Leu 420 425 430Glu Ser Gln Ala Ala
Leu Ala Val Leu Gly Pro Gln Thr Leu Gln Tyr 435 440 445Ser Tyr Thr
Pro Gln Leu Gln Asp Leu Asp Pro Leu Ala Gln Glu His 450 455 460Thr
Asp Ser Glu Glu Gly Pro Glu Glu Glu Pro Ser Thr Thr Leu Val465 470
475 480Asp Trp Asp Pro Gln Thr Gly Arg Leu Cys Ile Pro Ser Leu Ser
Ser 485 490 495Phe Asp Gln Asp Ser Glu Gly Cys Glu Pro Ser Glu Gly
Asp Gly Leu 500 505 510Gly Glu Glu Gly Leu Leu Ser Arg Leu Tyr Glu
Glu Pro Ala Pro Asp 515 520 525Arg Pro Pro Gly Glu Asn Glu Thr Tyr
Leu Met Gln Phe Met Glu Glu 530 535 540Trp Gly Leu Tyr Val Gln Met
Glu Asn545 55021911PRTHomo sapiensBCAN 21Met Ala Gln Leu Phe Leu
Pro Leu Leu Ala Ala Leu Val Leu Ala Gln1 5 10 15Ala Pro Ala Ala Leu
Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp 20 25 30Arg Ala Phe Arg
Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val 35 40 45Leu Gly Gly
Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro 50 55 60Pro Pro
Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr65 70 75
80Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Val
85 90 95Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro
Ala 100 105 110Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala Leu Ser
Glu Leu Arg 115 120 125Pro Asn Asp Ser Gly Ile Tyr Arg Cys Glu Val
Gln His Gly Ile Asp 130 135 140Asp Ser Ser Asp Ala Val Glu Val Lys
Val Lys Gly Val Val Phe Leu145 150 155 160Tyr Arg Glu Gly Ser Ala
Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln 165 170 175Glu Ala Cys Ala
Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu 180 185 190Tyr Ala
Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu 195 200
205Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala Cys
210 215 220Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly
Val Val225 230 235 240Asp Pro Asp Asp Leu Tyr Asp Val Tyr Cys Tyr
Ala Glu Asp Leu Asn 245 250 255Gly Glu Leu Phe Leu Gly Asp Pro Pro
Glu Lys Leu Thr Leu Glu Glu 260 265 270Ala Arg Ala Tyr Cys Gln Glu
Arg Gly Ala Glu Ile Ala Thr Thr Gly 275 280 285Gln Leu Tyr Ala Ala
Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly 290 295 300Trp Leu Ala
Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln305 310 315
320Arg Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro
325 330 335Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val
Tyr Cys 340 345 350Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu
Ala Ser Asn Pro 355 360 365Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu
Ala Ile Val Thr Val Thr 370 375 380Glu Thr Leu Glu Glu Leu Gln Leu
Pro Gln Glu Ala Thr Glu Ser Glu385 390 395 400Ser Arg Gly Ala Ile
Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly Gly 405 410 415Gly Ser Ser
Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu 420 425 430Glu
Phe Glu Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu 435 440
445Glu Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu
450 455 460Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala
Leu Trp465 470 475 480Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro
Glu Ala Ser Leu Pro 485 490 495Thr Glu Pro Ala Ala Gln Glu Lys Ser
Leu Ser Gln Ala Pro Ala Arg 500 505 510Ala Val Leu Gln Pro Gly Ala
Ser Pro Leu Pro Asp Gly Glu Ser Glu 515 520 525Ala Ser Arg Pro Pro
Arg Val His Gly Pro Pro Thr Glu Thr Leu Pro 530 535 540Thr Pro Arg
Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val545 550 555
560Glu Ala Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly
565 570 575Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly
Ala Pro 580 585 590Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr
Arg Glu Leu Glu 595 600 605Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr
Ala Pro Ala Gly Thr Ser 610 615 620Val Gln Ala Gln Pro Val Leu Pro
Thr Asp Ser Ala Ser Arg Gly Gly625 630 635 640Val Ala Val Val Pro
Ala Ser Gly Asp Cys Val Pro Ser Pro Cys His 645 650 655Asn Gly Gly
Thr Cys Leu Glu Glu Glu Glu Gly Val Arg Cys Leu Cys 660 665 670Leu
Pro Gly Tyr Gly Gly Asp Leu Cys Asp Val Gly Leu Arg Phe Cys 675 680
685Asn Pro Gly Trp Asp Ala Phe Gln Gly Ala Cys Tyr Lys His Phe Ser
690 695 700Thr Arg Arg Ser Trp Glu Glu Ala Glu Thr Gln Cys Arg Met
Tyr Gly705 710 715 720Ala His Leu Ala Ser Ile Ser Thr Pro Glu Glu
Gln Asp Phe Ile Asn 725 730 735Asn Arg Tyr Arg Glu Tyr Gln Trp Ile
Gly Leu Asn Asp Arg Thr Ile 740 745 750Glu Gly Asp Phe Leu Trp Ser
Asp Gly Val Pro Leu Leu Tyr Glu Asn 755 760 765Trp Asn Pro Gly Gln
Pro Asp Ser Tyr Phe Leu Ser Gly Glu Asn Cys 770 775 780Val Val Met
Val Trp His Asp Gln Gly Gln Trp Ser Asp Val Pro Cys785 790 795
800Asn Tyr His Leu Ser Tyr Thr Cys Lys Met Gly Leu Val Ser Cys Gly
805 810 815Pro Pro Pro Glu Leu Pro Leu Ala Gln Val Phe Gly Arg Pro
Arg Leu 820 825 830Arg Tyr Glu Val Asp Thr Val Leu Arg Tyr Arg Cys
Arg Glu Gly Leu 835 840 845Ala Gln Arg Asn Leu Pro Leu Ile Arg Cys
Gln Glu Asn Gly Arg Trp 850 855 860Glu Ala Pro Gln Ile Ser Cys Val
Pro Arg Arg Pro Ala Arg Ala Leu865 870 875 880His Pro Glu Glu Asp
Pro Glu Gly Arg Gln Gly Arg Leu Leu Gly Arg 885 890 895Trp Lys Ala
Leu Leu Ile Pro Pro Ser Ser Pro Met Pro Gly Pro 900 905
91022987PRTHomo sapiensEphB2R 22Met Ala Leu Arg Arg Leu Gly Ala Ala
Leu Leu Leu Leu Pro Leu Leu1 5 10 15Ala Ala Val Glu Glu Thr Leu Met
Asp Ser Thr Thr Ala Thr Ala Glu 20 25 30Leu Gly Trp Met Val His Pro
Pro Ser Gly Trp Glu Glu Val Ser Gly 35 40 45Tyr Asp Glu Asn Met Asn
Thr Ile Arg Thr Tyr Gln Val Cys Asn Val 50 55 60Phe Glu Ser Ser Gln
Asn Asn Trp Leu Arg Thr Lys Phe Ile Arg Arg65 70 75 80Arg Gly Ala
His Arg Ile His Val Glu Met Lys Phe Ser Val Arg Asp 85 90 95Cys Ser
Ser Ile Pro Ser Val Pro Gly Ser Cys Lys Glu Thr Phe Asn 100 105
110Leu Tyr Tyr Tyr Glu Ala Asp Phe Asp Ser Ala Thr Lys Thr Phe Pro
115 120 125Asn Trp Met Glu Asn Pro Trp Val Lys Val Asp Thr Ile Ala
Ala Asp 130 135 140Glu Ser Phe Ser Gln Val Asp Leu Gly Gly Arg Val
Met Lys Ile Asn145 150 155 160Thr Glu Val Arg Ser Phe Gly Pro Val
Ser Arg Ser Gly Phe Tyr Leu 165 170 175Ala Phe Gln Asp Tyr Gly Gly
Cys Met Ser Leu Ile Ala Val Arg Val 180 185 190Phe Tyr Arg Lys Cys
Pro Arg Ile Ile Gln Asn Gly Ala Ile Phe Gln 195 200 205Glu Thr Leu
Ser Gly Ala Glu Ser Thr Ser Leu Val Ala Ala Arg Gly 210 215 220Ser
Cys Ile Ala Asn Ala Glu Glu Val Asp Val Pro Ile Lys Leu Tyr225 230
235 240Cys Asn Gly Asp Gly Glu Trp Leu Val Pro Ile Gly Arg Cys Met
Cys 245 250 255Lys Ala Gly Phe Glu Ala Val Glu Asn Gly Thr Val Cys
Arg Gly Cys 260 265 270Pro Ser Gly Thr Phe Lys Ala Asn Gln Gly Asp
Glu Ala Cys Thr His 275 280 285Cys Pro Ile Asn Ser Arg Thr Thr Ser
Glu Gly Ala Thr Asn Cys Val 290 295 300Cys Arg Asn Gly Tyr Tyr Arg
Ala Asp Leu Asp Pro Leu Asp Met Pro305 310 315 320Cys Thr Thr Ile
Pro Ser Ala Pro Gln Ala Val Ile Ser Ser Val Asn 325 330 335Glu Thr
Ser Leu Met Leu Glu Trp Thr Pro Pro Arg Asp Ser Gly Gly 340 345
350Arg Glu Asp Leu Val Tyr Asn Ile Ile Cys Lys Ser Cys Gly Ser Gly
355 360 365Arg Gly Ala Cys Thr Arg Cys Gly Asp Asn Val Gln Tyr Ala
Pro Arg 370 375 380Gln Leu Gly Leu Thr Glu Pro Arg Ile Tyr Ile Ser
Asp Leu Leu Ala385 390 395 400His Thr Gln Tyr Thr Phe Glu Ile Gln
Ala Val Asn Gly Val Thr Asp 405 410 415Gln Ser Pro Phe Ser Pro Gln
Phe Ala Ser Val Asn Ile Thr Thr Asn 420 425 430Gln Ala Ala Pro Ser
Ala Val Ser Ile Met
His Gln Val Ser Arg Thr 435 440 445Val Asp Ser Ile Thr Leu Ser Trp
Ser Gln Pro Asp Gln Pro Asn Gly 450 455 460Val Ile Leu Asp Tyr Glu
Leu Gln Tyr Tyr Glu Lys Glu Leu Ser Glu465 470 475 480Tyr Asn Ala
Thr Ala Ile Lys Ser Pro Thr Asn Thr Val Thr Val Gln 485 490 495Gly
Leu Lys Ala Gly Ala Ile Tyr Val Phe Gln Val Arg Ala Arg Thr 500 505
510Val Ala Gly Tyr Gly Arg Tyr Ser Gly Lys Met Tyr Phe Gln Thr Met
515 520 525Thr Glu Ala Glu Tyr Gln Thr Ser Ile Gln Glu Lys Leu Pro
Leu Ile 530 535 540Ile Gly Ser Ser Ala Ala Gly Leu Val Phe Leu Ile
Ala Val Val Val545 550 555 560Ile Ala Ile Val Cys Asn Arg Arg Arg
Gly Phe Glu Arg Ala Asp Ser 565 570 575Glu Tyr Thr Asp Lys Leu Gln
His Tyr Thr Ser Gly His Met Thr Pro 580 585 590Gly Met Lys Ile Tyr
Ile Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glu 595 600 605Ala Val Arg
Glu Phe Ala Lys Glu Ile Asp Ile Ser Cys Val Lys Ile 610 615 620Glu
Gln Val Ile Gly Ala Gly Glu Phe Gly Glu Val Cys Ser Gly His625 630
635 640Leu Lys Leu Pro Gly Lys Arg Glu Ile Phe Val Ala Ile Lys Thr
Leu 645 650 655Lys Ser Gly Tyr Thr Glu Lys Gln Arg Arg Asp Phe Leu
Ser Glu Ala 660 665 670Ser Ile Met Gly Gln Phe Asp His Pro Asn Val
Ile His Leu Glu Gly 675 680 685Val Val Thr Lys Ser Thr Pro Val Met
Ile Ile Thr Glu Phe Met Glu 690 695 700Asn Gly Ser Leu Asp Ser Phe
Leu Arg Gln Asn Asp Gly Gln Phe Thr705 710 715 720Val Ile Gln Leu
Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys 725 730 735Tyr Leu
Ala Asp Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn 740 745
750Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu
755 760 765Ser Arg Phe Leu Glu Asp Asp Thr Ser Asp Pro Thr Tyr Thr
Ser Ala 770 775 780Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro
Glu Ala Ile Gln785 790 795 800Tyr Arg Lys Phe Thr Ser Ala Ser Asp
Val Trp Ser Tyr Gly Ile Val 805 810 815Met Trp Glu Val Met Ser Tyr
Gly Glu Arg Pro Tyr Trp Asp Met Thr 820 825 830Asn Gln Asp Val Ile
Asn Ala Ile Glu Gln Asp Tyr Arg Leu Pro Pro 835 840 845Pro Met Asp
Cys Pro Ser Ala Leu His Gln Leu Met Leu Asp Cys Trp 850 855 860Gln
Lys Asp Arg Asn His Arg Pro Lys Phe Gly Gln Ile Val Asn Thr865 870
875 880Leu Asp Lys Met Ile Arg Asn Pro Asn Ser Leu Lys Ala Met Ala
Pro 885 890 895Leu Ser Ser Gly Ile Asn Leu Pro Leu Leu Asp Arg Thr
Ile Pro Asp 900 905 910Tyr Thr Ser Phe Asn Thr Val Asp Glu Trp Leu
Glu Ala Ile Lys Met 915 920 925Gly Gln Tyr Lys Glu Ser Phe Ala Asn
Ala Gly Phe Thr Ser Phe Asp 930 935 940Val Val Ser Gln Met Met Met
Glu Asp Ile Leu Arg Val Gly Val Thr945 950 955 960Leu Ala Gly His
Gln Lys Lys Ile Leu Asn Ser Ile Gln Val Met Arg 965 970 975Ala Gln
Met Asn Gln Ile Gln Ser Val Glu Val 980 98523282PRTHomo
sapiensASLG659 23Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile
Ile Ser Ile Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile
Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr Val Thr Thr Val
Ala Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile Leu Ser Cys Thr
Phe Glu Pro Asp Ile Lys Leu 50 55 60Ser Asp Ile Val Ile Gln Trp Leu
Lys Glu Gly Val Leu Gly Leu Val65 70 75 80His Glu Phe Lys Glu Gly
Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95Phe Arg Gly Arg Thr
Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110Ala Ser Leu
Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125Lys
Cys Tyr Ile Ile Thr Ser Lys Gly Lys Lys Asn Ala Asn Leu Glu 130 135
140Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr
Asn145 150 155 160Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg
Trp Phe Pro Gln 165 170 175Pro Thr Val Val Trp Ala Ser Gln Val Asp
Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr Ser Phe Glu
Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val Ser Val Leu
Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220Cys Met Ile Glu
Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230 235 240Thr
Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser 245 250
255Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu
260 265 270Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys 275
28024123PRTHomo sapiensPSCA 24Met Lys Ala Val Leu Leu Ala Leu Leu
Met Ala Gly Leu Ala Leu Gln1 5 10 15Pro Gly Thr Ala Leu Leu Cys Tyr
Ser Cys Lys Ala Gln Val Ser Asn 20 25 30Glu Asp Cys Leu Gln Val Glu
Asn Cys Thr Gln Leu Gly Glu Gln Cys 35 40 45Trp Thr Ala Arg Ile Arg
Ala Val Gly Leu Leu Thr Val Ile Ser Lys 50 55 60Gly Cys Ser Leu Asn
Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly65 70 75 80Lys Lys Asn
Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly 85 90 95Ala His
Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala 100 105
110Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu 115 12025236PRTHomo
sapiensGEDA 25Met Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Met Leu1 5 10 15Pro Ala Gln Glu Ala Ala Lys Leu Tyr His Thr Asn
Tyr Val Arg Asn 20 25 30Ser Arg Ala Ile Gly Val Leu Trp Ala Ile Phe
Thr Ile Cys Phe Ala 35 40 45Ile Val Asn Val Val Cys Phe Ile Gln Pro
Tyr Trp Ile Gly Asp Gly 50 55 60Val Asp Thr Pro Gln Ala Gly Tyr Phe
Gly Leu Phe His Tyr Cys Ile65 70 75 80Gly Asn Gly Phe Ser Arg Glu
Leu Thr Cys Arg Gly Ser Phe Thr Asp 85 90 95Phe Ser Thr Leu Pro Ser
Gly Ala Phe Lys Ala Ala Ser Phe Phe Ile 100 105 110Gly Leu Ser Met
Met Leu Ile Ile Ala Cys Ile Ile Cys Phe Thr Leu 115 120 125Phe Phe
Phe Cys Asn Thr Ala Thr Val Tyr Lys Ile Cys Ala Trp Met 130 135
140Gln Leu Thr Ser Ala Ala Cys Leu Val Leu Gly Cys Met Ile Phe
Pro145 150 155 160Asp Gly Trp Asp Ser Asp Glu Val Lys Arg Met Cys
Gly Glu Lys Thr 165 170 175Asp Lys Tyr Thr Leu Gly Ala Cys Ser Val
Arg Trp Ala Tyr Ile Leu 180 185 190Ala Ile Ile Gly Ile Leu Asp Ala
Leu Ile Leu Ser Phe Leu Ala Phe 195 200 205Val Leu Gly Asn Arg Gln
Asp Ser Leu Met Ala Glu Glu Leu Lys Ala 210 215 220Glu Asn Lys Val
Leu Leu Ser Gln Tyr Ser Leu Glu225 230 23526184PRTHomo
sapiensBAFF-R 26Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala
Pro Ala Pro1 5 10 15Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu
Val Arg His Cys 20 25 30Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro
Lys Pro Ala Gly Ala 35 40 45Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln
Pro Gln Glu Ser Val Gly 50 55 60Ala Gly Ala Gly Glu Ala Ala Leu Pro
Leu Pro Gly Leu Leu Phe Gly65 70 75 80Ala Pro Ala Leu Leu Gly Leu
Ala Leu Val Leu Ala Leu Val Leu Val 85 90 95Gly Leu Val Ser Trp Arg
Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser 100 105 110Ser Ala Glu Ala
Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp 115 120 125Lys Val
Ile Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala 130 135
140Trp Pro Pro Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro Gly His
Ser145 150 155 160Val Pro Val Pro Ala Thr Glu Leu Gly Ser Thr Glu
Leu Val Thr Thr 165 170 175Lys Thr Ala Gly Pro Glu Gln Gln
18027847PRTHomo sapiensCD22 27Met His Leu Leu Gly Pro Trp Leu Leu
Leu Leu Val Leu Glu Tyr Leu1 5 10 15Ala Phe Ser Asp Ser Ser Lys Trp
Val Phe Glu His Pro Glu Thr Leu 20 25 30Tyr Ala Trp Glu Gly Ala Cys
Val Trp Ile Pro Cys Thr Tyr Arg Ala 35 40 45Leu Asp Gly Asp Leu Glu
Ser Phe Ile Leu Phe His Asn Pro Glu Tyr 50 55 60Asn Lys Asn Thr Ser
Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr65 70 75 80Lys Asp Gly
Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly 85 90 95Asp Lys
Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu Asn 100 105
110Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr Glu Lys Trp
115 120 125Met Glu Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro
Pro His 130 135 140Ile Gln Leu Pro Pro Glu Ile Gln Glu Ser Gln Glu
Val Thr Leu Thr145 150 155 160Cys Leu Leu Asn Phe Ser Cys Tyr Gly
Tyr Pro Ile Gln Leu Gln Trp 165 170 175Leu Leu Glu Gly Val Pro Met
Arg Gln Ala Ala Val Thr Ser Thr Ser 180 185 190Leu Thr Ile Lys Ser
Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro 195 200 205Gln Trp Ser
His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala 210 215 220Asp
Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys His225 230
235 240Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp Ala Ile Val
Arg 245 250 255Glu Gly Asp Ser Val Thr Met Thr Cys Glu Val Ser Ser
Ser Asn Pro 260 265 270Glu Tyr Thr Thr Val Ser Trp Leu Lys Asp Gly
Thr Ser Leu Lys Lys 275 280 285Gln Asn Thr Phe Thr Leu Asn Leu Arg
Glu Val Thr Lys Asp Gln Ser 290 295 300Gly Lys Tyr Cys Cys Gln Val
Ser Asn Asp Val Gly Pro Gly Arg Ser305 310 315 320Glu Glu Val Phe
Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val 325 330 335Gln Ile
Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu 340 345
350Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp Tyr His
355 360 365Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys Val His
Ile Pro 370 375 380Lys Ile Leu Pro Trp His Ala Gly Thr Tyr Ser Cys
Val Ala Glu Asn385 390 395 400Ile Leu Gly Thr Gly Gln Arg Gly Pro
Gly Ala Glu Leu Asp Val Gln 405 410 415Tyr Pro Pro Lys Lys Val Thr
Thr Val Ile Gln Asn Pro Met Pro Ile 420 425 430Arg Glu Gly Asp Thr
Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn 435 440 445Pro Ser Val
Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu 450 455 460Pro
Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr465 470
475 480Thr Ile Ala Cys Ala Arg Cys Asn Ser Trp Cys Ser Trp Ala Ser
Pro 485 490 495Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp Val Arg
Val Arg Lys 500 505 510Ile Lys Pro Leu Ser Glu Ile His Ser Gly Asn
Ser Val Ser Leu Gln 515 520 525Cys Asp Phe Ser Ser Ser His Pro Lys
Glu Val Gln Phe Phe Trp Glu 530 535 540Lys Asn Gly Arg Leu Leu Gly
Lys Glu Ser Gln Leu Asn Phe Asp Ser545 550 555 560Ile Ser Pro Glu
Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser 565 570 575Ile Gly
Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala 580 585
590Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met Glu
595 600 605Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn Pro
Pro Val 610 615 620Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln Ser
Leu Pro His His625 630 635 640Ser Gln Lys Leu Arg Leu Glu Pro Val
Lys Val Gln His Ser Gly Ala 645 650 655Tyr Trp Cys Gln Gly Thr Asn
Ser Val Gly Lys Gly Arg Ser Pro Leu 660 665 670Ser Thr Leu Thr Val
Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Val 675 680 685Ala Val Gly
Leu Gly Ser Cys Leu Ala Ile Leu Ile Leu Ala Ile Cys 690 695 700Gly
Leu Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly705 710
715 720Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn Lys
Lys 725 730 735Val Arg Arg Ala Pro Leu Ser Glu Gly Pro His Ser Leu
Gly Cys Tyr 740 745 750Asn Pro Met Met Glu Asp Gly Ile Ser Tyr Thr
Thr Leu Arg Phe Pro 755 760 765Glu Met Asn Ile Pro Arg Thr Gly Asp
Ala Glu Ser Ser Glu Met Gln 770 775 780Arg Pro Pro Arg Thr Cys Asp
Asp Thr Val Thr Tyr Ser Ala Leu His785 790 795 800Lys Arg Gln Val
Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu 805 810 815Asp Glu
Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu 820 825
830Arg Pro Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His 835
840 84528226PRTHomo sapiensCD79A 28Met Pro Gly Gly Pro Gly Val Leu
Gln Ala Leu Pro Ala Thr Ile Phe1 5 10 15Leu Leu Phe Leu Leu Ser Ala
Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30Leu Trp Met His Lys Val
Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45Asp Ala His Phe Gln
Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60Thr Trp Trp Arg
Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe65 70 75 80Leu Gly
Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90 95Asn
Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105
110Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro
115 120 125Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn
Arg Ile 130 135 140Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala
Val Val Pro Gly145 150 155 160Thr Leu Leu Leu Phe Arg Lys Arg Trp
Gln Asn Glu Lys Leu Gly Leu 165 170 175Asp Ala Gly Asp Glu Tyr Glu
Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190Leu Asp Asp Cys Ser
Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205Thr Tyr Gln
Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220Lys
Pro22529372PRTHomo sapiensCXCR5 29Met Asn Tyr Pro Leu Thr Leu
Glu Met Asp Leu Glu Asn Leu Glu Asp1 5 10 15Leu Phe Trp Glu Leu Asp
Arg Leu Asp Asn Tyr Asn Asp Thr Ser Leu 20 25 30Val Glu Asn His Leu
Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser 35 40 45Phe Lys Ala Val
Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu 50 55 60Gly Val Ile
Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg65 70 75 80Gln
Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala 85 90
95Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser
100 105 110Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile
Ala Leu 115 120 125His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu Leu
Ala Cys Ile Ala 130 135 140Val Asp Arg Tyr Leu Ala Ile Val His Ala
Val His Ala Tyr Arg His145 150 155 160Arg Arg Leu Leu Ser Ile His
Ile Thr Cys Gly Thr Ile Trp Leu Val 165 170 175Gly Phe Leu Leu Ala
Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln 180 185 190Gly His His
Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn 195 200 205Gln
Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val 210 215
220Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val
Gly225 230 235 240Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro
Gln Arg Gln Lys 245 250 255Ala Val Arg Val Ala Ile Leu Val Thr Ser
Ile Phe Phe Leu Cys Trp 260 265 270Ser Pro Tyr His Ile Val Ile Phe
Leu Asp Thr Leu Ala Arg Leu Lys 275 280 285Ala Val Asp Asn Thr Cys
Lys Leu Asn Gly Ser Leu Pro Val Ala Ile 290 295 300Thr Met Cys Glu
Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met305 310 315 320Leu
Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu 325 330
335Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe
340 345 350Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala
Thr Ser 355 360 365Leu Thr Thr Phe 37030273PRTHomo sapiensHLA-DOB
30Met Gly Ser Gly Trp Val Pro Trp Val Val Ala Leu Leu Val Asn Leu1
5 10 15Thr Arg Leu Asp Ser Ser Met Thr Gln Gly Thr Asp Ser Pro Glu
Asp 20 25 30Phe Val Ile Gln Ala Lys Ala Asp Cys Tyr Phe Thr Asn Gly
Thr Glu 35 40 45Lys Val Gln Phe Val Val Arg Phe Ile Phe Asn Leu Glu
Glu Tyr Val 50 55 60Arg Phe Asp Ser Asp Val Gly Met Phe Val Ala Leu
Thr Lys Leu Gly65 70 75 80Gln Pro Asp Ala Glu Gln Trp Asn Ser Arg
Leu Asp Leu Leu Glu Arg 85 90 95Ser Arg Gln Ala Val Asp Gly Val Cys
Arg His Asn Tyr Arg Leu Gly 100 105 110Ala Pro Phe Thr Val Gly Arg
Lys Val Gln Pro Glu Val Thr Val Tyr 115 120 125Pro Glu Arg Thr Pro
Leu Leu His Gln His Asn Leu Leu His Cys Ser 130 135 140Val Thr Gly
Phe Tyr Pro Gly Asp Ile Lys Ile Lys Trp Phe Leu Asn145 150 155
160Gly Gln Glu Glu Arg Ala Gly Val Met Ser Thr Gly Pro Ile Arg Asn
165 170 175Gly Asp Trp Thr Phe Gln Thr Val Val Met Leu Glu Met Thr
Pro Glu 180 185 190Leu Gly His Val Tyr Thr Cys Leu Val Asp His Ser
Ser Leu Leu Ser 195 200 205Pro Val Ser Val Glu Trp Arg Ala Gln Ser
Glu Tyr Ser Trp Arg Lys 210 215 220Met Leu Ser Gly Ile Ala Ala Phe
Leu Leu Gly Leu Ile Phe Leu Leu225 230 235 240Val Gly Ile Val Ile
Gln Leu Arg Ala Gln Lys Gly Tyr Val Arg Thr 245 250 255Gln Met Ser
Gly Asn Glu Val Ser Arg Ala Val Leu Leu Pro Gln Ser 260 265
270Cys31422PRTHomo sapiensPurinergic receptor P2X ligand-gated ion
channel 5 31Met Gly Gln Ala Gly Cys Lys Gly Leu Cys Leu Ser Leu Phe
Asp Tyr1 5 10 15Lys Thr Glu Lys Tyr Val Ile Ala Lys Asn Lys Lys Val
Gly Leu Leu 20 25 30Tyr Arg Leu Leu Gln Ala Ser Ile Leu Ala Tyr Leu
Val Val Trp Val 35 40 45Phe Leu Ile Lys Lys Gly Tyr Gln Asp Val Asp
Thr Ser Leu Gln Ser 50 55 60Ala Val Ile Thr Lys Val Lys Gly Val Ala
Phe Thr Asn Thr Ser Asp65 70 75 80Leu Gly Gln Arg Ile Trp Asp Val
Ala Asp Tyr Val Ile Pro Ala Gln 85 90 95Gly Glu Asn Val Phe Phe Val
Val Thr Asn Leu Ile Val Thr Pro Asn 100 105 110Gln Arg Gln Asn Val
Cys Ala Glu Asn Glu Gly Ile Pro Asp Gly Ala 115 120 125Cys Ser Lys
Asp Ser Asp Cys His Ala Gly Glu Ala Val Thr Ala Gly 130 135 140Asn
Gly Val Lys Thr Gly Arg Cys Leu Arg Arg Glu Asn Leu Ala Arg145 150
155 160Gly Thr Cys Glu Ile Phe Ala Trp Cys Pro Leu Glu Thr Ser Ser
Arg 165 170 175Pro Glu Glu Pro Phe Leu Lys Glu Ala Glu Asp Phe Thr
Ile Phe Ile 180 185 190Lys Asn His Ile Arg Phe Pro Lys Phe Asn Phe
Ser Lys Ser Asn Val 195 200 205Met Asp Val Lys Asp Arg Ser Phe Leu
Lys Ser Cys His Phe Gly Pro 210 215 220Lys Asn His Tyr Cys Pro Ile
Phe Arg Leu Gly Ser Val Ile Arg Trp225 230 235 240Ala Gly Ser Asp
Phe Gln Asp Ile Ala Leu Glu Gly Gly Val Ile Gly 245 250 255Ile Asn
Ile Glu Trp Asn Cys Asp Leu Asp Lys Ala Ala Ser Glu Cys 260 265
270His Pro His Tyr Ser Phe Ser Arg Leu Asp Asn Lys Leu Ser Lys Ser
275 280 285Val Ser Ser Gly Tyr Asn Phe Arg Phe Ala Arg Tyr Tyr Arg
Asp Ala 290 295 300Ala Gly Val Glu Phe Arg Thr Leu Met Lys Ala Tyr
Gly Ile Arg Phe305 310 315 320Asp Val Met Val Asn Gly Lys Gly Ala
Phe Phe Cys Asp Leu Val Leu 325 330 335Ile Tyr Leu Ile Lys Lys Arg
Glu Phe Tyr Arg Asp Lys Lys Tyr Glu 340 345 350Glu Val Arg Gly Leu
Glu Asp Ser Ser Gln Glu Ala Glu Asp Glu Ala 355 360 365Ser Gly Leu
Gly Leu Ser Glu Gln Leu Thr Ser Gly Pro Gly Leu Leu 370 375 380Gly
Met Pro Glu Gln Gln Glu Leu Gln Glu Pro Pro Glu Ala Lys Arg385 390
395 400Gly Ser Ser Ser Gln Lys Gly Asn Gly Ser Val Cys Pro Gln Leu
Leu 405 410 415Glu Pro His Arg Ser Thr 42032359PRTHomo sapiensCD72
32Met Ala Glu Ala Ile Thr Tyr Ala Asp Leu Arg Phe Val Lys Ala Pro1
5 10 15Leu Lys Lys Ser Ile Ser Ser Arg Leu Gly Gln Asp Pro Gly Ala
Asp 20 25 30Asp Asp Gly Glu Ile Thr Tyr Glu Asn Val Gln Val Pro Ala
Val Leu 35 40 45Gly Val Pro Ser Ser Leu Ala Ser Ser Val Leu Gly Asp
Lys Ala Ala 50 55 60Val Lys Ser Glu Gln Pro Thr Ala Ser Trp Arg Ala
Val Thr Ser Pro65 70 75 80Ala Val Gly Arg Ile Leu Pro Cys Arg Thr
Thr Cys Leu Arg Tyr Leu 85 90 95Leu Leu Gly Leu Leu Leu Thr Cys Leu
Leu Leu Gly Val Thr Ala Ile 100 105 110Cys Leu Gly Val Arg Tyr Leu
Gln Val Ser Gln Gln Leu Gln Gln Thr 115 120 125Asn Arg Val Leu Glu
Val Thr Asn Ser Ser Leu Arg Gln Gln Leu Arg 130 135 140Leu Lys Ile
Thr Gln Leu Gly Gln Ser Ala Glu Asp Leu Gln Gly Ser145 150 155
160Arg Arg Glu Leu Ala Gln Ser Gln Glu Ala Leu Gln Val Glu Gln Arg
165 170 175Ala His Gln Ala Ala Glu Gly Gln Leu Gln Ala Cys Gln Ala
Asp Arg 180 185 190Gln Lys Thr Lys Glu Thr Leu Gln Ser Glu Glu Gln
Gln Arg Arg Ala 195 200 205Leu Glu Gln Lys Leu Ser Asn Met Glu Asn
Arg Leu Lys Pro Phe Phe 210 215 220Thr Cys Gly Ser Ala Asp Thr Cys
Cys Pro Ser Gly Trp Ile Met His225 230 235 240Gln Lys Ser Cys Phe
Tyr Ile Ser Leu Thr Ser Lys Asn Trp Gln Glu 245 250 255Ser Gln Lys
Gln Cys Glu Thr Leu Ser Ser Lys Leu Ala Thr Phe Ser 260 265 270Glu
Ile Tyr Pro Gln Ser His Ser Tyr Tyr Phe Leu Asn Ser Leu Leu 275 280
285Pro Asn Gly Gly Ser Gly Asn Ser Tyr Trp Thr Gly Leu Ser Ser Asn
290 295 300Lys Asp Trp Lys Leu Thr Asp Asp Thr Gln Arg Thr Arg Thr
Tyr Ala305 310 315 320Gln Ser Ser Lys Cys Asn Lys Val His Lys Thr
Trp Ser Trp Trp Thr 325 330 335Leu Glu Ser Glu Ser Cys Arg Ser Ser
Leu Pro Tyr Ile Cys Glu Met 340 345 350Thr Ala Phe Arg Phe Pro Asp
35533661PRTHomo sapiensLY64 33Met Ala Phe Asp Val Ser Cys Phe Phe
Trp Val Val Leu Phe Ser Ala1 5 10 15Gly Cys Lys Val Ile Thr Ser Trp
Asp Gln Met Cys Ile Glu Lys Glu 20 25 30Ala Asn Lys Thr Tyr Asn Cys
Glu Asn Leu Gly Leu Ser Glu Ile Pro 35 40 45Asp Thr Leu Pro Asn Thr
Thr Glu Phe Leu Glu Phe Ser Phe Asn Phe 50 55 60Leu Pro Thr Ile His
Asn Arg Thr Phe Ser Arg Leu Met Asn Leu Thr65 70 75 80Phe Leu Asp
Leu Thr Arg Cys Gln Ile Asn Trp Ile His Glu Asp Thr 85 90 95Phe Gln
Ser His His Gln Leu Ser Thr Leu Val Leu Thr Gly Asn Pro 100 105
110Leu Ile Phe Met Ala Glu Thr Ser Leu Asn Gly Pro Lys Ser Leu Lys
115 120 125His Leu Phe Leu Ile Gln Thr Gly Ile Ser Asn Leu Glu Phe
Ile Pro 130 135 140Val His Asn Leu Glu Asn Leu Glu Ser Leu Tyr Leu
Gly Ser Asn His145 150 155 160Ile Ser Ser Ile Lys Phe Pro Lys Asp
Phe Pro Ala Arg Asn Leu Lys 165 170 175Val Leu Asp Phe Gln Asn Asn
Ala Ile His Tyr Ile Ser Arg Glu Asp 180 185 190Met Arg Ser Leu Glu
Gln Ala Ile Asn Leu Ser Leu Asn Phe Asn Gly 195 200 205Asn Asn Val
Lys Gly Ile Glu Leu Gly Ala Phe Asp Ser Thr Val Phe 210 215 220Gln
Ser Leu Asn Phe Gly Gly Thr Pro Asn Leu Ser Val Ile Phe Asn225 230
235 240Gly Leu Gln Asn Ser Thr Thr Gln Ser Leu Trp Leu Gly Thr Phe
Glu 245 250 255Asp Ile Asp Asp Glu Asp Ile Ser Ser Ala Met Leu Lys
Gly Leu Cys 260 265 270Glu Met Ser Val Glu Ser Leu Asn Leu Gln Glu
His Arg Phe Ser Asp 275 280 285Ile Ser Ser Thr Thr Phe Gln Cys Phe
Thr Gln Leu Gln Glu Leu Asp 290 295 300Leu Thr Ala Thr His Leu Lys
Gly Leu Pro Ser Gly Met Lys Gly Leu305 310 315 320Asn Leu Leu Lys
Lys Leu Val Leu Ser Val Asn His Phe Asp Gln Leu 325 330 335Cys Gln
Ile Ser Ala Ala Asn Phe Pro Ser Leu Thr His Leu Tyr Ile 340 345
350Arg Gly Asn Val Lys Lys Leu His Leu Gly Val Gly Cys Leu Glu Lys
355 360 365Leu Gly Asn Leu Gln Thr Leu Asp Leu Ser His Asn Asp Ile
Glu Ala 370 375 380Ser Asp Cys Cys Ser Leu Gln Leu Lys Asn Leu Ser
His Leu Gln Thr385 390 395 400Leu Asn Leu Ser His Asn Glu Pro Leu
Gly Leu Gln Ser Gln Ala Phe 405 410 415Lys Glu Cys Pro Gln Leu Glu
Leu Leu Asp Leu Ala Phe Thr Arg Leu 420 425 430His Ile Asn Ala Pro
Gln Ser Pro Phe Gln Asn Leu His Phe Leu Gln 435 440 445Val Leu Asn
Leu Thr Tyr Cys Phe Leu Asp Thr Ser Asn Gln His Leu 450 455 460Leu
Ala Gly Leu Pro Val Leu Arg His Leu Asn Leu Lys Gly Asn His465 470
475 480Phe Gln Asp Gly Thr Ile Thr Lys Thr Asn Leu Leu Gln Thr Val
Gly 485 490 495Ser Leu Glu Val Leu Ile Leu Ser Ser Cys Gly Leu Leu
Ser Ile Asp 500 505 510Gln Gln Ala Phe His Ser Leu Gly Lys Met Ser
His Val Asp Leu Ser 515 520 525His Asn Ser Leu Thr Cys Asp Ser Ile
Asp Ser Leu Ser His Leu Lys 530 535 540Gly Ile Tyr Leu Asn Leu Ala
Ala Asn Ser Ile Asn Ile Ile Ser Pro545 550 555 560Arg Leu Leu Pro
Ile Leu Ser Gln Gln Ser Thr Ile Asn Leu Ser His 565 570 575Asn Pro
Leu Asp Cys Thr Cys Ser Asn Ile His Phe Leu Thr Trp Tyr 580 585
590Lys Glu Asn Leu His Lys Leu Glu Gly Ser Glu Glu Thr Thr Cys Ala
595 600 605Asn Pro Pro Ser Leu Arg Gly Val Lys Leu Ser Asp Val Lys
Leu Ser 610 615 620Cys Gly Ile Thr Ala Ile Gly Ile Phe Phe Leu Ile
Val Phe Leu Leu625 630 635 640Leu Leu Ala Ile Leu Leu Phe Phe Ala
Val Lys Tyr Leu Leu Arg Trp 645 650 655Lys Tyr Gln His Ile
66034429PRTHomo sapiensFCRH1 34Met Leu Pro Arg Leu Leu Leu Leu Ile
Cys Ala Pro Leu Cys Glu Pro1 5 10 15Ala Glu Leu Phe Leu Ile Ala Ser
Pro Ser His Pro Thr Glu Gly Ser 20 25 30Pro Val Thr Leu Thr Cys Lys
Met Pro Phe Leu Gln Ser Ser Asp Ala 35 40 45Gln Phe Gln Phe Cys Phe
Phe Arg Asp Thr Arg Ala Leu Gly Pro Gly 50 55 60Trp Ser Ser Ser Pro
Lys Leu Gln Ile Ala Ala Met Trp Lys Glu Asp65 70 75 80Thr Gly Ser
Tyr Trp Cys Glu Ala Gln Thr Met Ala Ser Lys Val Leu 85 90 95Arg Ser
Arg Arg Ser Gln Ile Asn Val His Arg Val Pro Val Ala Asp 100 105
110Val Ser Leu Glu Thr Gln Pro Pro Gly Gly Gln Val Met Glu Gly Asp
115 120 125Arg Leu Val Leu Ile Cys Ser Val Ala Met Gly Thr Gly Asp
Ile Thr 130 135 140Phe Leu Trp Tyr Lys Gly Ala Val Gly Leu Asn Leu
Gln Ser Lys Thr145 150 155 160Gln Arg Ser Leu Thr Ala Glu Tyr Glu
Ile Pro Ser Val Arg Glu Ser 165 170 175Asp Ala Glu Gln Tyr Tyr Cys
Val Ala Glu Asn Gly Tyr Gly Pro Ser 180 185 190Pro Ser Gly Leu Val
Ser Ile Thr Val Arg Ile Pro Val Ser Arg Pro 195 200 205Ile Leu Met
Leu Arg Ala Pro Arg Ala Gln Ala Ala Val Glu Asp Val 210 215 220Leu
Glu Leu His Cys Glu Ala Leu Arg Gly Ser Pro Pro Ile Leu Tyr225 230
235 240Trp Phe Tyr His Glu Asp Ile Thr Leu Gly Ser Arg Ser Ala Pro
Ser 245 250 255Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Glu Glu
His Ser Gly 260 265 270Asn Tyr Ser Cys Glu Ala Asn Asn Gly Leu Gly
Ala Gln Arg Ser Glu 275 280 285Ala Val Thr Leu Asn Phe Thr Val Pro
Thr Gly Ala Arg Ser Asn His 290 295 300Leu Thr Ser Gly Val Ile Glu
Gly Leu Leu Ser Thr Leu Gly Pro Ala305 310 315 320Thr Val Ala Leu
Leu Phe Cys Tyr Gly Leu Lys Arg Lys Ile Gly Arg 325 330 335Arg Ser
Ala Arg Asp Pro Leu Arg Ser Leu Pro Ser Pro Leu Pro Gln 340 345
350Glu Phe Thr Tyr Leu Asn Ser Pro Thr Pro Gly Gln Leu Gln Pro Ile
355 360 365Tyr
Glu Asn Val Asn Val Val Ser Gly Asp Glu Val Tyr Ser Leu Ala 370 375
380Tyr Tyr Asn Gln Pro Glu Gln Glu Ser Val Ala Ala Glu Thr Leu
Gly385 390 395 400Thr His Met Glu Asp Lys Val Ser Leu Asp Ile Tyr
Ser Arg Leu Arg 405 410 415Lys Ala Asn Ile Thr Asp Val Asp Tyr Glu
Asp Ala Met 420 42535977PRTHomo sapiensIRTA2 35Met Leu Leu Trp Val
Ile Leu Leu Val Leu Ala Pro Val Ser Gly Gln1 5 10 15Phe Ala Arg Thr
Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro Trp Thr 20 25 30Thr Val Phe
Gln Gly Glu Arg Val Thr Leu Thr Cys Lys Gly Phe Arg 35 40 45Phe Tyr
Ser Pro Gln Lys Thr Lys Trp Tyr His Arg Tyr Leu Gly Lys 50 55 60Glu
Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu Glu Val Gln Glu Ser65 70 75
80Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser Pro Leu Ser Ser Pro Val
85 90 95His Leu Asp Phe Ser Ser Ala Ser Leu Ile Leu Gln Ala Pro Leu
Ser 100 105 110Val Phe Glu Gly Asp Ser Val Val Leu Arg Cys Arg Ala
Lys Ala Glu 115 120 125Val Thr Leu Asn Asn Thr Ile Tyr Lys Asn Asp
Asn Val Leu Ala Phe 130 135 140Leu Asn Lys Arg Thr Asp Phe His Ile
Pro His Ala Cys Leu Lys Asp145 150 155 160Asn Gly Ala Tyr Arg Cys
Thr Gly Tyr Lys Glu Ser Cys Cys Pro Val 165 170 175Ser Ser Asn Thr
Val Lys Ile Gln Val Gln Glu Pro Phe Thr Arg Pro 180 185 190Val Leu
Arg Ala Ser Ser Phe Gln Pro Ile Ser Gly Asn Pro Val Thr 195 200
205Leu Thr Cys Glu Thr Gln Leu Ser Leu Glu Arg Ser Asp Val Pro Leu
210 215 220Arg Phe Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu Gly
Trp Ser225 230 235 240Leu Ser Pro Asn Phe Gln Ile Thr Ala Met Trp
Ser Lys Asp Ser Gly 245 250 255Phe Tyr Trp Cys Lys Ala Ala Thr Met
Pro His Ser Val Ile Ser Asp 260 265 270Ser Pro Arg Ser Trp Ile Gln
Val Gln Ile Pro Ala Ser His Pro Val 275 280 285Leu Thr Leu Ser Pro
Glu Lys Ala Leu Asn Phe Glu Gly Thr Lys Val 290 295 300Thr Leu His
Cys Glu Thr Gln Glu Asp Ser Leu Arg Thr Leu Tyr Arg305 310 315
320Phe Tyr His Glu Gly Val Pro Leu Arg His Lys Ser Val Arg Cys Glu
325 330 335Arg Gly Ala Ser Ile Ser Phe Ser Leu Thr Thr Glu Asn Ser
Gly Asn 340 345 350Tyr Tyr Cys Thr Ala Asp Asn Gly Leu Gly Ala Lys
Pro Ser Lys Ala 355 360 365Val Ser Leu Ser Val Thr Val Pro Val Ser
His Pro Val Leu Asn Leu 370 375 380Ser Ser Pro Glu Asp Leu Ile Phe
Glu Gly Ala Lys Val Thr Leu His385 390 395 400Cys Glu Ala Gln Arg
Gly Ser Leu Pro Ile Leu Tyr Gln Phe His His 405 410 415Glu Asp Ala
Ala Leu Glu Arg Arg Ser Ala Asn Ser Ala Gly Gly Val 420 425 430Ala
Ile Ser Phe Ser Leu Thr Ala Glu His Ser Gly Asn Tyr Tyr Cys 435 440
445Thr Ala Asp Asn Gly Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu
450 455 460Ser Ile Thr Val Pro Val Ser His Pro Val Leu Thr Leu Ser
Ser Ala465 470 475 480Glu Ala Leu Thr Phe Glu Gly Ala Thr Val Thr
Leu His Cys Glu Val 485 490 495Gln Arg Gly Ser Pro Gln Ile Leu Tyr
Gln Phe Tyr His Glu Asp Met 500 505 510Pro Leu Trp Ser Ser Ser Thr
Pro Ser Val Gly Arg Val Ser Phe Ser 515 520 525Phe Ser Leu Thr Glu
Gly His Ser Gly Asn Tyr Tyr Cys Thr Ala Asp 530 535 540Asn Gly Phe
Gly Pro Gln Arg Ser Glu Val Val Ser Leu Phe Val Thr545 550 555
560Val Pro Val Ser Arg Pro Ile Leu Thr Leu Arg Val Pro Arg Ala Gln
565 570 575Ala Val Val Gly Asp Leu Leu Glu Leu His Cys Glu Ala Pro
Arg Gly 580 585 590Ser Pro Pro Ile Leu Tyr Trp Phe Tyr His Glu Asp
Val Thr Leu Gly 595 600 605Ser Ser Ser Ala Pro Ser Gly Gly Glu Ala
Ser Phe Asn Leu Ser Leu 610 615 620Thr Ala Glu His Ser Gly Asn Tyr
Ser Cys Glu Ala Asn Asn Gly Leu625 630 635 640Val Ala Gln His Ser
Asp Thr Ile Ser Leu Ser Val Ile Val Pro Val 645 650 655Ser Arg Pro
Ile Leu Thr Phe Arg Ala Pro Arg Ala Gln Ala Val Val 660 665 670Gly
Asp Leu Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Ser Pro 675 680
685Ile Leu Tyr Trp Phe Tyr His Glu Asp Val Thr Leu Gly Lys Ile Ser
690 695 700Ala Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr
Thr Glu705 710 715 720His Ser Gly Ile Tyr Ser Cys Glu Ala Asp Asn
Gly Pro Glu Ala Gln 725 730 735Arg Ser Glu Met Val Thr Leu Lys Val
Ala Val Pro Val Ser Arg Pro 740 745 750Val Leu Thr Leu Arg Ala Pro
Gly Thr His Ala Ala Val Gly Asp Leu 755 760 765Leu Glu Leu His Cys
Glu Ala Leu Arg Gly Ser Pro Leu Ile Leu Tyr 770 775 780Arg Phe Phe
His Glu Asp Val Thr Leu Gly Asn Arg Ser Ser Pro Ser785 790 795
800Gly Gly Ala Ser Leu Asn Leu Ser Leu Thr Ala Glu His Ser Gly Asn
805 810 815Tyr Ser Cys Glu Ala Asp Asn Gly Leu Gly Ala Gln Arg Ser
Glu Thr 820 825 830Val Thr Leu Tyr Ile Thr Gly Leu Thr Ala Asn Arg
Ser Gly Pro Phe 835 840 845Ala Thr Gly Val Ala Gly Gly Leu Leu Ser
Ile Ala Gly Leu Ala Ala 850 855 860Gly Ala Leu Leu Leu Tyr Cys Trp
Leu Ser Arg Lys Ala Gly Arg Lys865 870 875 880Pro Ala Ser Asp Pro
Ala Arg Ser Pro Pro Asp Ser Asp Ser Gln Glu 885 890 895Pro Thr Tyr
His Asn Val Pro Ala Trp Glu Glu Leu Gln Pro Val Tyr 900 905 910Thr
Asn Ala Asn Pro Arg Gly Glu Asn Val Val Tyr Ser Glu Val Arg 915 920
925Ile Ile Gln Glu Lys Lys Lys His Ala Val Ala Ser Asp Pro Arg His
930 935 940Leu Arg Asn Lys Gly Ser Pro Ile Ile Tyr Ser Glu Val Lys
Val Ala945 950 955 960Ser Thr Pro Val Ser Gly Ser Leu Phe Leu Ala
Ser Ser Ala Pro His 965 970 975Arg
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